Microfluidic sequencing methods

ABSTRACT

Integrated systems, apparatus, software, and methods for performing biochemical analysis, including DNA sequencing, genomic screening, purification of nucleic acids and other biological components and drug screening are provided. Microfluidic devices, systems and methods for using these devices and systems for performing a wide variety of fluid operations are provided. The devices and systems of are used in performing fluid operations which require a large number of iterative, successive or parallel fluid manipulations, in a microscale, or sealed and readily automated format.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of provisional patentapplication U.S. Pat. No. 60/068311, entitled “Closed Loop BiochemicalAnalyzer” by Knapp, filed Dec. 19, 1997. The subject application is alsoa continuation-in-part of Ser. No. 08/835,101 by Knapp et al. filed Apr.4, 1997 (converted to a provisional application by filing a petitionunder 37 C.F.R. §§ 1.53(C) and 1.17(a) on Jan. 20, 1998), entitled“Microfluidic Devices and Systems for Performing Integrated FluidOperations.” Both of these applications are incorporated herein byreference in their entirety for all purposes.

FIELD OF THE INVENTION

[0002] This application relates to apparatus, methods and integratedsystems for detecting molecular interactions. The apparatus comprisemicroscale devices for moving and mixing small fluid volumes. Thesystems are capable of performing integrated manipulation and analysisin a variety of biological, biochemical and chemical experiments,including, e.g., DNA sequencing.

BACKGROUND OF THE INVENTION

[0003] Manipulating fluidic reagents and assessing the results ofreagent interactions are central to chemical and biological science.Manipulations include mixing fluidic reagents, assaying productsresulting from such mixtures, and separation or purification of productsor reagents and the like. Assessing the results of reagent interactionscan include autoradiography, spectroscopy, microscopy, photography, massspectrometry, nuclear magnetic resonance and many other techniques forobserving and recording the results of mixing reagents. A singleexperiment can involve literally hundreds of fluidic manipulations,product separations, result recording processes and data compilation andintegration steps. Fluidic manipulations are performed using a widevariety of laboratory equipment, including various fluid heatingdevices, fluidic mixing devices, centrifugation equipment, moleculepurification apparatus, chromatographic machinery, gel electrophoreticequipment and the like. The effects of mixing fluidic reagents aretypically assessed by additional equipment relating to detection,visualization or recording of an event to be assayed, such asspectrophotometers, autoradiographic equipment, microscopes, gelscanners, computers and the like.

[0004] Because analysis of even simple chemical, biochemical, orbiological phenomena requires many different types of laboratoryequipment, the modern laboratory is complex, large and expensive. Inaddition, because so many different types of equipment are used in evenconceptually simple experiments such as DNA sequencing, it has notgenerally been practical to integrate different types of equipment toimprove automation. The need for a laboratory worker to physicallyperform many aspects of laboratory science imposes sharp limits on thenumber of experiments which a laboratory can perform, and increases theundesirable exposure of laboratory workers to toxic or radioactivereagents. In addition, results are often analyzed manually, with theselection of subsequent experiments related to initial experimentsrequiring consideration by a laboratory worker, severely limiting thethroughput of even repetitive experimentation.

[0005] In an attempt to increase laboratory throughput and to decreaseexposure of laboratory workers to reagents, various strategies have beenperformed. For example, robotic introduction of fluids onto microtiterplates is commonly performed to speed mixing of reagents and to enhanceexperimental throughput. More recently, microscale devices for highthroughput mixing and assaying of small fluid volumes have beendeveloped. For example, U.S. Ser. No. 08/761,575 entitled “HighThroughput Screening Assay Systems in Microscale Fluidic Devices” byParce et al. provides pioneering technology related to microscalefluidic devices, especially including electrokinetic devices. Thedevices are generally suitable for assays relating to the interaction ofbiological and chemical species, including enzymes and substrates,ligands and ligand binders, receptors and ligands, antibodies andantibody ligands, as well as many other assays. Because the devicesprovide the ability to mix fluidic reagents and assay mixing results ina single continuous process, and because minute amounts of reagents canbe assayed, these microscale devices represent a fundamental advance forlaboratory science.

[0006] In the electrokinetic microscale devices provided by Parce et al.above, an appropriate fluid is flowed into a microchannel etched in asubstrate having functional groups present at the surface. The groupsionize when the surface is contacted with an aqueous solution. Forexample, where the surface of the channel includes hydroxyl functionalgroups at the surface, e.g., as in glass substrates, protons can leavethe surface of the channel and enter the fluid. Under such conditions,the surface possesses a net negative charge, whereas the fluid willpossess an excess of protons, or positive charge, particularly localizednear the interface between the channel surface and the fluid. Byapplying an electric field along the length of the channel, cations willflow toward the negative electrode. Movement of the sheath of positivelycharged species in the fluid pulls the solvent with them.

[0007] One time consuming process is titration of biological andbiochemical assay components into the dynamic range of an assay. Forexample, because enzyme activities vary from lot to lot, it is necessaryto perform a titration of enzyme and substrate concentrations todetermine optimum reaction conditions. Similarly, diagnostic assaysrequire titration of unknown concentrations of components so that theassay can be performed using appropriate concentrations of components.Thus, even before performing a typical diagnostic assay, severalnormalization steps need to be performed with assay components.

[0008] Another labor intensive laboratory process is the selection oflead compounds in drug screening assays. Various approaches to screeningfor lead compounds are reviewed by Janda (1994) Proc. Natl. Acad. Sci.USA 91(10779-10785); Blondelle (1995) Trends Anal. Chem 14:83-91; Chenet al. (1995) Angl. Chem. Int. Engl. 34:953-960; Ecker et al. (1995)Bio/Technology 13:351-360; Gordon et al. (1994) J. Med. Chem.37:1385-1401 and Gallop et al. (1994) J. Med. Chem. 37:1233-1251.Improvements in screening have been developed by combining one or moresteps in the screening process, e.g., affinity capillaryelectrophoresis-mass spectrometry for combinatorial library screening(Chu et al. (1996) J. Am. Chem. Soc. 118:7827-7835). However, thesehigh-throughput screening methods do not provide an integrated way ofselecting a second assay or screen based upon the results of a firstassay or screen. Thus, results from one assay are not automatically usedto focus subsequent experimentation and experimental design stillrequires a large input of labor by the user.

[0009] Another particularly labor intensive biochemical series oflaboratory fluidic manipulations is nucleic acid sequencing. EfficientDNA sequencing technology is central to the development of thebiotechnology industry and basic biological research. Improvements inthe efficiency and speed of DNA sequencing are needed to keep pace withthe demands for DNA sequence information. The Human Genome Project, forexample, has set a goal of dramatically increasing the efficiency,cost-effectiveness and throughput of DNA sequencing techniques. See,e.g., Collins, and Galas (1993) Science 262:43-46.

[0010] Most DNA sequencing today is carried out by chain terminationmethods of DNA sequencing. The most popular chain termination methods ofDNA sequencing are variants of the dideoxynucleotide mediated chaintermination method of Sanger. See, Sanger et al. (1977) Proc. Nat. Acad.Sci., USA 74:5463-5467. For a simple introduction to dideoxy sequencing,see, Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (Supplement 37, current through 1997)(Ausubel), Chapter 7. Four color sequencing is described in U.S. Pat.No. 5,171,534. Thousands of laboratories employ dideoxynucleotide chaintermination techniques. Commercial kits containing the reagents mosttypically used for these methods of DNA sequencing are available andwidely used.

[0011] In addition to the Sanger methods of chain termination, new PCRexonuclease digestion methods have also been proposed for DNAsequencing. Direct sequencing of PCR generated amplicons by selectivelyincorporating boronated nuclease resistant nucleotides into theamplicons during PCR and digestion of the amplicons with a nuclease toproduce sized template fragments has been proposed (Porter et al. (1997)Nucleic Acids Research 25(8):1611-1617). In the methods, 4 PCR reactionson a template are performed, in each of which one of the nucleotidetriphosphates in the PCR reaction mixture is partially substituted witha 2′ deoxynucleoside 5′-α[P-borano]-triphosphate. The boronatednucleotide is stocastically incorporated into PCR products at varyingpositions along the PCR amplicon in a nested set of PCR fragments of thetemplate. An exonuclease which is blocked by incorporated boronatednucleotides is used to cleave the PCR amplicons. The cleaved ampliconsare then separated by size using polyacrylamide gel electrophoresis,providing the sequence of the amplicon. An advantage of this method isthat it requires fewer biochemical manipulations than performingstandard Sanger-style sequencing of PCR amplicons.

[0012] Other sequencing methods which reduce the number of stepsnecessary for template preparation and primer selection have beendeveloped. One proposed variation on sequencing technology involves theuse of modular primers for use in PCR and DNA sequencing. For example,Ulanovsky and co-workers have described the mechanism of the modularprimer effect (Beskin et al. (1995) Nucleic Acids Research23(15):2881-2885) in which short primers of 5-6 nucleotides canspecifically prime a template-dependent polymerase enzyme for templatedependent nucleic acid synthesis. A modified version of the use of themodular primer strategy, in which small nucleotide primers arespecifically elongated for use in PCR to amplify and sequence templatenucleic acids has also been described. The procedure is referred to asDNA sequencing using differential extension with nucleotide subsets(DENS). See, Raja et al. (1997) Nucleic Acids Research 25(4):800-805.

[0013] In addition to enzymatic and other chain termination sequencingmethods, sequencing by hybridization to complementary oligonucleotideshas been proposed, e.g., in U.S. Pat. No. 5,202,231, to Drmanac et al.and, e.g., in Drmanac et al. (1989) Genomics 4:114-128. Chemicaldegradation sequencing methods are also well known and still in use;see, Maxam and Gilbert (1980) in Grossman and Moldave (eds.) AcademicPress, New York, Methods in Enzymology 65:499-560.

[0014] Improvements in methods for generating sequencing templates havealso been developed. DNA sequencing typically involves three steps: i)making suitable templates for the regions to be sequenced; ii) runningsequencing reactions for electrophoresis and iii) assessing the resultsof the reaction. The latter steps are sometimes automated by use oflarge and very expensive workstations and autosequencers. The first stepoften requires careful experimental design and laborious DNAmanipulation such as the construction of nested deletion mutants. See,Griffin, H. G. and Griffin, A. M. (1993) DNA sequencing protocols,Humana Press, New Jersey. Alternatively, random “shot-gun” sequencingmethods, are sometimes used to make templates, in which randomlyselected sub clones, which may or may not have overlapping sequenceinformation, are randomly sequenced. The sequences of the sub clones arecompiled to produce an ordered sequence. This procedures eliminatescomplicated DNA manipulations; however, the method is inherentlyinefficient because many recombinant clones must be sequenced due to therandom nature of the procedure. Because of the labor intensive nature ofsequencing, the repetitive sequencing of many individual clonesdramatically reduces the throughput of these sequencing systems.

[0015] Recently, Hagiwara and Curtis (1996) Nucleic Acids Research24(12):2460-2461 developed a “long distance sequencer” PCR protocol forgenerating overlapping nucleic acids from very large clones tofacilitate sequencing, and methods of amplifying and tagging theoverlapping nucleic acids into suitable sequencing templates. Themethods can be used in conjunction with shotgun sequencing techniques toimprove the efficiency of shotgun methods.

[0016] Although improvements in robotic manipulation of fluidic reagentsand miniaturization of laboratory equipment have been made, and althoughparticular biochemical processes such as DNA sequencing and drugscreening are very well developed, there still exists a need foradditional techniques and apparatus for mixing and assaying fluidicreagents, for integration of such systems and for reduction of thenumber of manipulations required to perform biochemical manipulationssuch as drug screening and DNA sequencing. Ideally, these new apparatuswould be useful with, and compatible to, established biochemicalprotocols. This invention provides these and many other features.

SUMMARY OF THE INVENTION

[0017] This invention provides apparatus, systems and methods forintegrated manipulation and analysis of fluidic reagents. The integratedfeatures provide very high throughput methods of assessing biochemicalcomponents and performing biochemical manipulations. A wide variety ofreagents and products are suitably assessed, including libraries ofchemical or biological compounds or components, nucleic acid templates,PCR reaction products, and the like. In the integrated systems it ispossible to use the results of a first reaction or set of reactions toselect appropriate reagents, reactants, products, or the like, foradditional analysis. For example, the results of a first sequencingreaction can be used to select primers, templates or the like foradditional sequencing, or to select related families of compounds forscreening in high-throughput assay methods. These primers or templatesare then accessed by the system and the process continues.

[0018] In one aspect, the invention provides integrated methods ofanalyzing and manipulating sample materials for fluidic analysis. In themethods, an integrated microfluidic system including a microfluidicdevice is provided. The device has at least a first reaction channel andat least a first reagent introduction channel, typically etched,machined, printed, or otherwise manufactured in or on a substrate.Optionally, the device can have a second reaction channel and/or reagentintroduction channel, a third reaction channel and/or reagentintroduction channel or the like, up to and including hundreds or eventhousands of reaction and/or reagent introduction channels. The reactionchannel and reagent introduction channels are in fluid communication,i.e., fluid can flow between the channels under selected conditions. Thedevice has a material transport system for controllably transporting amaterial through and among the reagent introduction channel and reactionchannel. For example, the material transport system can includeelectrokinetic, electroosmotic, electrophoretic or other fluidmanipulation aspects (micro-pumps and microvalves, fluid switches, fluidgates, etc.) which permit controlled movement and mixing of fluids. Thedevice also has a fluidic interface in fluid communication with thereagent introduction channel. Such fluidic interfaces optionally includecapillaries, channels, pins, pipettors, electropipettors, or the like,for moving fluids, and optionally further include microscopic,spectroscopic, fluid separatory or other aspects. The fluidic interfacesamples a plurality of reagents or mixtures of reagents from a pluralityof sources of reagents or mixtures of reagents and introduces thereagents or mixtures of reagents into the reagent introduction channel.Essentially any number of reagents or reagent mixtures can be introducedby the fluidic interface, depending on the desired application. Becausemicrofluidic manipulations are performed in a partially or fully sealedenvironment, contamination and fluidic evaporation in the systems areminimized.

[0019] In the methods, a first reagent from the plurality of sources ofreagent or mixtures of reagents is selected. A first sample material andthe first reagent or mixture of reagents is introduced into the firstreaction channel, whereupon the first sample material and the firstreagent or mixture of reagents react. This reaction can take a varietyof different forms depending on the nature of the reagents. For example,where the reagents bind to one another, such as where the reagents arean antibody or cell receptor and a ligand, or an amino acid and abinding ligand, the reaction results in a bound component such as abound ligand. Where the reagents are sequencing reagents, a primerextension product results from the reaction. Where the reagents includeenzymes and enzyme substrates, a modified form of the substratetypically results. Where two reacting chemical reagents are mixed, athird product chemical typically results.

[0020] In the methods, a reaction product of the first sample materialand the first reagent or mixture of reagents is analyzed. This analysiscan take any of a variety of forms, depending on the application. Forexample, where the product is a primer extension product, the analysiscan take the form of separating reactants by size, detecting the sizedreactants and translating the resulting information to give the sequenceof a template nucleic acid. Similarly, because microscale fluidicdevices of the invention are optionally suitable for heating and coolinga reaction, a PCR reaction utilizing PCR reagents (thermostablepolymerase, nucleotides, templates, primers, buffers and the like) canbe performed and the PCR reagents detected. Where the reaction resultsin the formation of a new product, such as an enzyme-substrate product,a chemical species, or an immunological component such as a boundligand, the product is typically detected by any of a variety ofdetection techniques, including autoradiography, microscopy,spectroscopy, or the like.

[0021] Based upon the reaction product, a second reagent or mixture ofreagents is selected and a second sample material is assessed. Forexample, where the product is a DNA sequence, a sequencing primer and/ortemplate for extension of available sequence information is selected.Where the product is a new product such as those above, an appropriatesecond component such as an enzyme, ligand, antibody, receptor molecule,chemical, or the like, is selected to further test the binding orreactive characteristics of an analyzed material. The second reagent ormixture of reagents is introduced into the first reaction channel, oroptionally into a second (or third or fourth . . . or nth) reactionchannel in the microfluidic device. The second sample material and thesecond reagent or mixture of reagents react, forming a new product,which is analyzed as above. The results of the analysis can serve as thebasis for the selection and analysis of additional reactants for similarsubsequent analysis. The second sample material, reagents, or mixturesof reagents can comprise the same or different materials. For example, asingle type of DNA template is optionally sequenced in several serialreactions. Alternatively, completing a first sequencing reaction, asoutlined above, serves as the basis for selecting additional templates(e.g., overlapping clones, PCR amplicons, or the like).

[0022] Accordingly, in a preferred aspect, the invention providesmethods of sequencing a nucleic acid. In the methods, the biochemicalcomponents of a sequencing reaction (e.g., a target nucleic acid, afirst and optionally, second sequencing primer, a polymerase (optionallyincluding thermostable polymerases for use in PCR), dNTPs, and ddNTPs)are mixed in a microfluidic device under conditions permitting targetdependent polymerization of the dNTPs. Polymerization products areseparated in the microfluidic device to provide a sequence of the targetnucleic acid. Typically, sequencing information acquired by this methodis used to select additional sequencing primers and/or templates, andthe process is reiterated. Generally, a second sequencing primer isselected based upon the sequence of the target nucleic acid and thesecond sequencing primer is mixed with the target nucleic acid in amicrofluidic device under conditions permitting target dependentelongation of the selected second sequencing primer, thereby providingpolymerization products which are separated by size in the microfluidicdevice to provide further sequence of the target nucleic acid. Asdiscussed above, the systems for mixing the biochemical sequencingcomponents, separating the reaction products, and assessing the resultsof the sequencing reaction are integrated into a single system.

[0023] In one integrated sequencing system, methods of sequencing atarget nucleic acid are provided in which an integrated microfluidicsystem comprising a microfluidic device is utilized in the sequencingmethod. The integrated microfluidic device has at least a firstsequencing reaction channel and at least a first sequencing reagentintroduction channel, the sequencing reaction channel and sequencingreagent introduction channel being in fluid communication. Theintegrated microfluidic system also has a material transport system forcontrollably transporting sequencing reagents through the sequencingreagent introduction channel and sequencing reaction channel and afluidic interface in fluid communication with the sequencing reagentintroduction channel for sampling a plurality of sequencing reagents, ormixtures of sequencing reagents, from a plurality of sources ofsequencing reagents or mixtures of sequencing reagents and introducingthe sequencing reagents or mixtures of sequencing reagents into thesequence reagent introduction channel. As discussed above, the interfaceoptionally includes capillaries, pins, pipettors and the like. In themethod, a first sequencing primer sequence complementary to a firstsubsequence of a first target nucleic acid sequence is introduced intothe sequence reagent introduction channel. The first primer ishybridized to the first subsequence and the first primer is extendedwith a polymerase enzyme along the length of the target nucleic acidsequence to form a first extension product that is complementary to thefirst subsequence and a second subsequence of the target nucleic acid.The sequence of the first extension product is determined and, basedupon the sequence of the first extension product, a second primersequence complementary to a second subsequence of the target nucleicacid sequence is selected, hybridized and extended as above.

[0024] In the sequence methods herein, it is sometimes advantageous tohave select sequencing primers from a large set of sequencing primers,rather than synthesizing primers to match a particular target nucleicacid. For example, 5 or 6-mer primers can be made to hybridizespecifically to a target, e.g., where the primers are modular andhybridize to a single region of a nucleic acid. All possible 5 or 6 merscan be synthesized for selection in the methods herein, or any subset of5 or 6 mers can also be selected. In some embodiments, the primers aretransferred to the microfluidic apparatus, e.g., by a capillary, anelectropipettor, or using sipping technology, from a microtiter plate orfrom and array of oligos. In other embodiments, the primers are locatedon a region of a microfluidic device, chip or other substrate.

[0025] An advantage of these sequencing methods is that theydramatically increase the speed with which sequencing reactions can beperformed. An entire sequencing reaction, separation of sequencingproducts and sequence generation can be performed in less than an hour,often less than 30 minutes, generally less than 15 minutes, sometimesless than 10 minutes and occasionally less than 5 minutes.

[0026] The present invention provides integrated systems and apparatusfor performing the sequencing methods herein. In one embodiment, theinvention provides a sequencing apparatus. The apparatus has a topportion, a bottom portion and an interior portion. The interior portionhas at least two intersecting channels (and often tens, hundreds, orthousands of intersecting channels), wherein at least one of the twointersecting channels has at least one cross sectional dimension betweenabout 0.1 μm and 500 μm. A preferred embodiment of the inventionincludes an electrokinetic fluid direction system for moving asequencing reagent through at least one of the two intersectingchannels. The apparatus further includes a mixing zone fluidly connectedto the at least two intersecting channels for mixing the sequencingreagents, and a size separation zone fluidly connected to the mixingzone for separating sequencing products by size, thereby providing thesequence of a target nucleic acid. Optionally, the apparatus has asequence detector for reading the sequence of the target nucleic acid.In one preferred embodiment, the apparatus has a set of wells forreceiving reagents such as primer sets for use in the apparatus. In oneembodiment, the apparatus has at least 4,096 wells fluidly connected tothe at least two intersecting channels. Alternatively, the apparatus caninclude a substrate (matrix, or membrane) with primers located on thesubstrate. Often, the primers will be dried in spots on the substrate.In this embodiment, the apparatus will typically include anelectropipettor which has a tip designed to re-hydrate a selected spotcorresponding to a dried primer, and for electrophoretic transport ofthe rehydrated primer to an analysis region in the microfluidic device(i.e., a component of the microfluidic device which includes a reactionchannel). Thus, in a preferred embodiment, the device will include asubstrate such as a membrane having, e.g., 4,096 spots (i.e., allpossible 6-mer primers). Similarly, components in diagnostic or drugscreening assays can be stored in the well or membrane format forintroduction into the analysis region of the device. Arrays of nucleicacids, proteins and other compounds are also used in a similar manner.

[0027] In another embodiment, the invention provides systems fordetermining a sequence of nucleotides in a target nucleic acid sequence.The system includes a microfluidic device having a body structure withat least a first mixing or analysis channel, and at least a first probeintroduction channel disposed therein, the analysis channel intersectingand being in fluid communication with the probe introduction channel.The system includes a source of the target nucleic acid sequence influid communication with the analysis channel and a plurality ofseparate sources of oligonucleotide probes in fluid communication withthe probe introduction channel, each of the plurality of separatesources containing an oligonucleotide probe having a differentnucleotide sequence of length n. Typically, all or essentially all(i.e., most, i.e., at least about 70%, typically 90% or more) of thepossible oligonucleotides of a given length are included, although asubset of all possible oligonucleotides can also be used. The systemalso includes a sampling system for separately transporting a volume ofeach of the oligonucleotide probes from the sources of oligonucleotideprobes to the probe introduction channel and injecting each of theoligonucleotide probes into the analysis channel to contact the targetnucleic acid sequence and a detection system for identifying whethereach oligonucleotide probe hybridizes with the target nucleic acidsequence.

[0028] Methods of using the system for sequencing by hybridization toperfectly matched probes are also provided. In these methods, a targetnucleic acid is flowed into the analysis channel and a plurality ofextension probes are separately injected into the analysis channel,whereupon the extension probes contact the target nucleic acid sequence.In the method, a first subsequence of nucleotides in the target nucleicacid is typically known, and each of the plurality of extension probeshas a first sequence portion that is perfectly complementary to at leasta portion of the first subsequence, and an extension portion thatcorresponds to a portion of the target nucleic acid sequence adjacent tothe target subsequence, the extension portion having a length n, andcomprising all possible nucleotide sequences of length n, wherein n isbetween 1 and 4 inclusive. A sequence of nucleotides is identifiedadjacent the target subsequence, based upon which of the plurality ofextension probes perfectly hybridizes with the target nucleic acidsequence.

BRIEF DESCRIPTION OF THE DRAWING

[0029]FIG. 1 depicts a graph of florescence signal of intercalating dyefor lambda genomic DNA.

[0030]FIG. 2 depicts a thermocycler channel with varying widths forperforming, e.g., PCR.

[0031]FIG. 3 depicts a top view of a non-thermal amplificationapparatus.

[0032]FIG. 4A-4D depicts a top view of a reaction and separationapparatus and output data from the apparatus.

[0033]FIG. 5A depicts a top view of an apparatus for discriminatingnucleic acids based on sequencing; 5B depicts an optional separationchannel.

[0034]FIG. 6A-6B depict alternate technologies for flowing driedreagents from a substrate into a microfluidic apparatus; 6A depicts anelectropipettor with a cup region; 6B depicts an electrokineticinterface which spans a membrane having the dried reagents.

[0035]FIG. 7A-7D depict serial to parallel conversion strategies.

[0036]FIG. 8 depicts a top view of a serial to parallel converter.

[0037]FIG. 9 depicts a top view of a serial to parallel converter.

[0038]FIG. 10 depicts a top view of a serial to parallel converter.

[0039]FIG. 11 depicts a top view of a serial to parallel converter.

[0040]FIG. 12 depicts a block diagram of a control system as connectedto a microfluidic device.

[0041]FIG. 13 is a top view of an integrated microfluidic device havinga storage substrate in the same plane as an analysis substrate.

[0042]FIG. 14 is a top view of an integrated microfluidic devices havinga storage substrate in a plane different from an analysis substrate.

[0043]FIG. 15 is a top view of a microfluidic substrate having anintegrated electropipettor.

[0044]FIG. 16 is a top view of a microfluidic substrate having anintegrated electropipettor in the form of a capillary tube.

[0045]FIG. 17 is a top view of a microfluidic substrate having anintegrated electropipettor with serpentine channel geometry useful forelectrophoresis.

[0046]FIG. 18 is a top view of an integrated microfluidic deviceincorporating a microtiter dish.

[0047]FIG. 19 is a flowchart outlining some of the software processingsteps performed by a computer in an integrated system of the invention.

[0048]FIG. 20 is a schematic of an integrated system for sequencingnucleic acids.

[0049]FIG. 21 is a top view of a microchip of the invention.

[0050]FIG. 22 is an electropherogram for an assay.

[0051]FIG. 23 is an electropherogram for an assay in which white bloodcells are electrophoresed.

DEFINITIONS

[0052] An “integrated microfluidic system” is a microfluidic system inwhich a plurality of fluidic operations are performed. In oneembodiment, the results of a first reaction in the microfluidic systemare used to select reactants or other reagents for a second reaction orassay. The system will typically include a microfluidic substrate, and afluidic interface for sampling reactants or other components. A detectorand a computer are often included for detecting reaction products andfor recording, selecting, facilitating and monitoring reactions in themicrofluidic substrate.

[0053] A “microfluidic device” is an apparatus or component of anapparatus having microfluidic reaction channels and/or chambers.Typically, at least one reaction channel or chamber will have at leastone cross-sectional dimension between about 0.1 μm and about 500 μm.

[0054] A “reaction channel” is a channel (in any form, including aclosed channel, a capillary, a trench, groove or the like) on or in amicrofluidic substrate (a chip, bed, wafer, laminate, or the like havingmicrofluidic channels) in which two or more components are mixed. Thechannel will have at least one region with a cross sectional dimensionof between about 0.1 μm and about 500 μm.

[0055] A “reagent channel” is a channel (in any form, including a closedchannel, a capillary, a trench, groove or the like) on or in amicrofluidic substrate (a chip, bed, wafer, laminate, or the like havingmicrofluidic channels) through which components are transported(typically suspended or dissolved in a fluid). The channel will have atleast one region with a cross sectional dimension of between about 0.1μm and about 500 μm.

[0056] A “material transport system” is a system for moving componentsalong or through microfluidic channels. Exemplar transport systemsinclude electrokinetic, electroosmotic, and electrophoretic systems(e.g., electrodes in fluidly connected wells having a coupled currentand/or voltage controller), as well as micro-pump and valve systems.

[0057] A “fluidic interface” in the context of a microfluidic substrateis a component for transporting materials into or out of the substrate.The interface can include, e.g., an electropipettor, capillaries,channels, pins, pipettors, sippers or the like for moving fluids intothe microfluidic substrate.

[0058] The overall function, i.e., intended goal, of the devices,systems and methods of the invention are generally referred to as“fluidic operations.” For example, where a device's intended function isto screen a sample against a panel of antigens, the entire screen isreferred to as a single fluidic operation. Similarly, the fluidicoperation of a device intended to amplify nucleic acids is thecompletion of the amplification process, including all of the numerousmelting, annealing extension cycles. However, the individual steps ofthe overall fluidic operation are generally referred to as a “fluidmanipulation.” In the screening example, the combination or mixture of aportion of the sample with a solution containing a single antigen wouldconstitute a fluid manipulation. Similarly, in the amplificationexample, each separate reagent addition step required for each separatecycling step would constitute a single fluid manipulation. In manycases, the fluids utilized in the microfluidic devices and methods ofthe invention are referred to as reactants to denote their ability toundergo a chemical reaction, either alone, or when combined with anotherreactive fluid or composition. It will be readily apparent that thephrases “fluidic operation” and “fluid manipulation” encompass a widevariety of such manipulations for carrying out a variety of chemical,biological and biochemical reactions, either entirely fluid based orincorporating a non-fluid element, e.g., cells, solid supports,catalysts, etc., including, reagent additions, combinations,extractions, filtrations, purifications, and the like.

[0059] A “sequencing primer” is an oligonucleotide primer which is canbe extended with a polymerase in the presence of a template andappropriate reagents (dNTPs, etc).

DETAILED DESCRIPTION

[0060] High throughput manipulation and analysis of fluidic reagents isdesirable for a variety of applications, including nucleic acidsequencing, screening of chemical or biological libraries, purificationof molecules of interest, amplification of nucleic acids and the like.The present invention provides apparatus, systems and methods fordramatically increasing the speed and simplicity of screening,manipulating and assessing fluidic reagents, reagent mixtures, reactionproducts (including the products of DNA sequencing reactions) and thelike. The invention provides integrated systems for performing a varietyof chemical, biochemical and biological experiments and other fluidicoperations, including PCR, DNA sequencing, integrated or sequentialscreening of chemical or biological libraries, and the like. Althoughthe microfluidic systems of the invention are generally described interms of the performance of chemical, biochemical or biologicalreactions separations, incubations and the like, it will be understoodthat, as fluidic systems having general applicability, these systems canhave a wide variety of different uses, e.g., as metering or dispensingsystems in both biological and nonbiological applications.

[0061] In the methods of the prior art, most fluidic operations aregenerally performable at the bench scale, e.g., involving reagentvolumes ranging from 10 μl to 1 or more liters. However, the performanceof large numbers of iterative, successive or parallel fluidmanipulations at the bench scale potentially includes a number ofassociated problems. For example, when performed manually, repetitivetasks, e.g., fluid measurement and addition, are often plagued by errorsand mistakes, which often result in the overall failure of the overalloperation. Similarly, iterative or successive processing of small fluidsamples often results in substantial yield problems, e.g., from loss ofmaterial during incomplete fluid transfers, i.e., resulting fromincomplete transfer of fluid volumes, adsorption of materials onreaction vessels, pipettes and the like. These problems cansubstantially reduce the accuracy and reproducibility of a particularprocess performed manually, or at the bench scale. Further, in fluidicoperations that employ large numbers of parallel fluid manipulations,while the individual separate reactions are not overly cumbersome, thelogistics of coordinating and carrying out each of the parallelmanipulations can become unmanageable. Additionally, the costs,complexity and space requirements of equipment for facilitating theseoperations, e.g., robotics, creates further difficulties in performingthese types of operations.

[0062] In addition to the above, where reagent costs are substantial,even at the low end of the volume spectrum, a particular fluidicoperation involving numerous iterative or parallel reagent additions,can be commercially impracticable from a cost standpoint. Further, asreagent volumes become smaller and smaller, errors in measurement becomemore and more problematic. By performing iterative, successive orparallel fluid manipulations in microfluidic devices that are partiallysealed and automatable, the above-described problems of measurement andfluid transfer errors, reagent costs, equipment costs and spacerequirements are alleviated.

[0063] Accordingly, in one aspect, the present invention providesmicrofluidic devices, systems and methods that are particularly usefulin performing fluid operations that require a large number of iterativefluid manipulations. By “iterative fluid manipulations” is meant themovement and/or direction, incubation/reaction, separation or detectionof discrete volumes of fluid, typically in a serial format ororientation, in a repetitive fashion, i.e., performing the same type ofmanipulation on multiple separate samples, diluting a particular sample,etc., typically while varying one or more parameter in each series ofreactions. When performed at bench scales, iterative fluid manipulationsbecome relatively cumbersome as the number of repetitions becomesgreater, resulting in a substantial increase in the likelihood of errorsin measurement or the like, and requiring massive labor inputs as a userhas to select which parameters or reagents to vary in each successiveoperation. As such, the systems and devices of the present invention areparticularly useful in performing such iterative fluid manipulations,e.g., which require performance of a particular fluid manipulationgreater than about 10 times, typically greater than about 20 times,preferably greater than about 50 times and often greater than about 100times. In particularly preferred aspects, such fluid manipulations arerepeated between about 10 and 100 times or between about 100 and 1000times.

[0064] The present invention, therefore, provides microfluidic systemsand methods that are useful for performing a wide variety of differentfluidic operations, i.e., chemical, biochemical or biological reactions,incubations, separations, and the like, which, when performed bypreviously known methods, would be difficult or cumbersome, either interms of time, space, labor and/or costs. In particular, the systems ofthe present invention permit the performance of a wide variety offluidic operations without requiring large amounts of space, expensivereagents and/or equipment, or excessive time and labor costs.Specifically, as microfluidic devices are employed, the methods andsystems of the invention utilize less space and have smaller reagentrequirements. In addition, because these microfluidic systems areautomatable and partially sealed, they can reduce the amount of humaninvolvement in these manipulations, saving labor and eliminating many ofthe areas that are prone to human error, e.g., contamination,measurement errors, loss of materials and the like. A powerful newadditional aspect of the present invention is the ability of theapparatus, systems and methods to select components of iterative assaysbased upon the results of previous assays.

[0065] In its simplest embodiment, iterative fluid manipulation includesthe repeated movement, direction or delivery of a discrete volume of aparticular reagent to or through a particular reaction chamber orchannel. In more complex embodiments, such iterative fluid manipulationsinclude the apportioning of larger fluid volumes into smaller, discretefluid volumes, which includes the aliquoting of a given sample among anumber of separate reaction chambers or channels, or the taking ofaliquots from numerous discrete fluids, e.g., samples, to deliver thesealiquots to the same or different reaction chambers or channels.

[0066] In another, similar aspect, the devices, systems and methods ofthe invention are useful in performing fluidic operations that require alarge number of successive fluid manipulations, i.e., in performing anumber of preparative and analytical reactions or operations on a givensample. By “successive fluid manipulations” is generally meant a fluidicoperation that involves the successive treatment of a given fluid samplevolume, i.e., combination/reaction with reactants, incubation,purification/separation, analysis of products, and the like. Wheresuccessive fluid manipulations are performed at the bench scale, e.g.,the performance of numerous, different manipulations on a particularsample such as combination with reagents, incubation, separation anddetection, such manipulations can also become cumbersome as the numberof steps increases, as with each step, the possibility of introducing anerror into the operation or experiment increases. This complexity, andthe consequent increased possibility of errors increases substantiallyas the number of samples to be passed through the operation increases.Thus, the devices or systems of the present invention are alsoparticularly useful in performing fluidic operations which requiresuccessive fluid manipulations of a given sample or fluid of interest,e.g., more than 2 steps or different manipulations, typically greaterthan 5 steps or different manipulations, preferably greater than 10steps or different fluid manipulations. The systems are also useful andreadily capable of performing fluidic operations that include greaterthan 20, 50, 100, 1000 steps or different fluid manipulations on a givenfluid volume.

[0067] In a related, but alternate aspect, the devices, systems andmethods of the invention are useful in performing fluidic operationsthat require a large number of parallel fluid manipulations, i.e., toscreen biological samples, screen test compounds for drug discovery,e.g., as set forth in U.S. patent application Ser. Nos. 08/671,987 and08/671,986, both filed Jun. 28, 1996 and incorporated herein byreference. To carry out these operations, a substrate will typicallyemploy an array of parallel channels and/or channel networks,interconnected by one or more common channels. Fluids required for thesubject reaction, e.g., samples or reagents, are directed along one ormore of the common channels, and are delivered to each of the parallelchannels.

[0068] As used herein, “parallel fluid manipulations” means thesubstantially concurrent movement and/or direction, incubation/reaction,separation or detection of discrete fluid volumes to a plurality ofparallel channels and/or channel networks, or chambers of a microfluidicdevice, i.e., greater than about 10 distinct parallel channels orchambers, typically greater than 20 distinct channels or chambers,preferably greater than about 50 distinct channels or chambers, andoften greater than about 100 distinct channels or chambers. As usedherein, the term “parallel” refers to the ability to concomitantly orsubstantially concurrently process two or more separate fluid volumes,and does not necessarily denote a specific channel or chamber structureor layout.

[0069] Ultra high-throughput analysis systems are provided, for examplefor performing nucleic acids-based diagnostic and sequencingapplications, e.g., in a reference laboratory setting. The systemtypically has several components: a specimen and reagents handlingsystem; an “operating system” for processing integrated microchipexperimentation steps; application-specific analysis devices (optionallyreferred in this application e.g., as “LabChips™” (LabChip™ is atrademark of Caliper Technologies, Corp., Palo Alto Calif.); afluorescence-based signal detection system, and multiple softwarecomponents that allow the user to interact with the system, and runprocessing steps, interpret data, and report results.

Application to Sequencing Projects

[0070] In a preferred aspect, the invention provides a closed loopdevice for determining the entire sequence of an unknown DNA molecule ofinterest by iteratively sequencing sub regions of the molecule ofinterest. In one aspect, oligonucleotides are chosen from a pool ofpossible sequencing primers upon determination of an initial portion ofthe DNA sequence. With iterative utilization of this strategy, it ispossible to walk through an entire sequence without synthesizing newprimers.

[0071] “Primer walking” is a standard strategy for determining thesequence of an unknown DNA. For example, a portion of an unsequenced DNAcloned into a plasmid can be sequenced using a primer complementary to aportion of the plasmid, and extending the sequencing reaction into theunknown region of the DNA with a template dependent polymerase. However,standard electrophoretic analysis of the sequencing reaction only allowsresolution of a few hundred nucleotides. Once the sequence of a fewhundred nucleotides is determined, a second primer is synthesized to becomplementary to a portion of the sequenced region, and the reaction isrepeated, giving a new sequence which yields an additional few hundrednucleotides. Although the process is conceptually simple, it is alsovery labor intensive and time consuming for large nucleotide sequences.For example, sequencing a Yeast Artificial Chromosome (YAC) clone of amodest 100,000 bases using this serial primer walking fashion wouldrequire at least about 300-1,000 individual reactions, with acorresponding number of primer syntheses. It should also be noted thateach of these primer syntheses typically produces thousands of times asmuch primer as needed for the particular sequencing reaction,dramatically increasing the cost of sequencing.

[0072] The present invention simplifies the standard primer walkingstrategy by modifying, automating and integrating each part of primerwalking into a single integrated system. In the methods of theinvention, all of the mixing and analysis steps are performed with anintegrated system, and all primer synthetic steps are preferablyavoided. In brief, a template nucleic acid is selected and introducedinto a reaction channel in a microfluidic (generally electroosmotic)device of the invention. The template is optionally amplified, e.g., byintroducing PCR or LCR reagents into the channel and performing cyclesof heating and cooling on the template. Alternatively, e.g., where thesource of template is from an abundant sequence such as a cloned nucleicacid, further amplification can be unnecessary. In addition toamplification procedures, a PCR nuclease chain termination procedure canalso be used for direct sequencing in the methods of the invention.Porter et al. (1997) Nucleic Acids Research 25(8):1611-1617 describe thebiochemistry of PCR chain termination methods.

[0073] Sequencing reagents are added to the template nucleic acid and asequencing reaction is performed appropriate to the particular reactionin use. Many appropriate reactions are known, with the Sanger dideoxychain termination method being the most common. See, Sanger et al.(1977) Proc. Nat. Acad. Sci., USA 74:5463-5467. The primer used to primesynthesis is typically selected from a pre-synthesized set of nucleicacid primers, preferably a set including many or all of the primers fora particular primer length. In a preferred aspect, modular primers areused.

[0074] After the sequencing reaction is run, the products are separatedby size and/or charge in an analysis region of the microfluidic device.As discussed herein, the devices of the invention can be used toelectrophoretically separate macromolecules by size and/or charge. Theseparated products are detected, often as they pass a detector (nucleicacids are typically labeled with radioactive nucleotides orfluorophores; accordingly appropriate detectors includespectrophotometers, fluorescent detectors, microscopes (e.g., forfluorescent microscopy) and scintillation counting devices). Detectionof the size separated products is used to compile sequence informationfor the region being sequenced. A computer is used to select a secondprimer from the pre-synthesized primer set which hybridizes to thesequenced region, and the process is iteratively repeated with thesecond primer, leading to sequencing of a second region, selection of athird primer hybridizing to the second region, etc.

Providing DNA Templates for Sequencing

[0075] The integrated systems of the invention are useful for sequencinga wide variety of nucleic acid constructs. Essentially any DNA templatecan be sequenced, with the selection of the nucleic acid to be sequenceddepending upon the construct in hand by the sequencer. Thus, an initialstep in the methods of the invention is the selection or production of atemplate nucleic acid to be sequenced.

[0076] Many methods of making recombinant ribo and deoxyribo nucleicacids, including recombinant plasmids, recombinant lambda phage,cosmids, yeast artificial chromosomes (YACs), P1 artificial chromosomes,Bacterial Artificial Chromosomes (BACs), and the like are known. Thesequencing of large nucleic acid templates is advantageously performedby the present methods, systems and apparatus, because an entire nucleicacid can be sequenced by primer walking along the length of the templatein several rapid cycles of sequencing.

Cloning Templates or other Targets for use in the Methods, Apparatus andSystems of the invention

[0077] Examples of appropriate cloning techniques for making nucleicacids, and instructions sufficient to direct persons of skill throughmost standard cloning and other template preparation exercises are foundin Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods inEnzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger);Sambrook et al. (1989) Molecular Cloning-A Laboratory Manual (2nd ed.)Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY,(Sambrook); and Current Protocols in Molecular Biology, F. M. Ausubel etal., eds., Current Protocols, a joint venture between Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc., (1997, supplement 37)(Ausubel). Basic procedures for cloning and other aspects of molecularbiology and underlying theoretical considerations are also found inLewin (1995) Genes V Oxford University Press Inc., NY (Lewin); andWatson et al. (1992) Recombinant DNA Second Edition Scientific AmericanBooks, NY. Product information from manufacturers of biological reagentsand experimental equipment also provide information useful in knownbiological methods. Such manufacturers include the Sigma ChemicalCompany (Saint Louis, Mo.); New England Biolabs (Beverly, Mass.); R&Dsystems (Minneapolis, Minn.); Pharmacia LKB Biotechnology (Piscataway,N.J.); CLONTECH Laboratories, Inc. (Palo Alto, Calif.); ChemGenes Corp.,(Waltham Mass.) Aldrich Chemical Company (Milwaukee, Wis.); GlenResearch, Inc. (Sterling, Va.); GIBCO BRL Life Technologies, Inc.(Gaithersberg, Md.); Fluka Chemica-Biochemika Analytika (Fluka ChemieAG, Buchs, Switzerland); Invitrogen (San Diego, Calif.); Perkin Elmer(Foster City, Calif.); and Strategene; as well as many other commercialsources known to one of skill.

[0078] In one aspect, the generation of large nucleic acids is useful inpracticing the invention. It will be appreciated that such templates areparticularly useful in some aspects where the methods and devices of theinvention are used to sequence large regions of DNA, e.g., for genomicstypes of applications. An introduction to large clones such as YACs,BACs, PACs and MACs as artificial chromosomes is provided by Monaco andLarin (1994) Trends Biotechnol 12 (7): 280-286.

[0079] The construction of nucleic acid libraries of template nucleicacids is described in the above references. YACs and YAC libraries arefurther described in Burke et al. (1987) Science 236:806-812. Griddedlibraries of YACs are described in Anand et al. (1989) Nucleic AcidsRes. 17, 3425-3433, and Anand et al. (1990) Nucleic Acids Res. Riley(1990) 18:1951-1956 Nucleic Acids Res. 18(10): 2887-2890 and thereferences therein describe cloning of YACs and the use of vectorettesin conjunction with YACs. See also, Ausubel, chapter 13. Cosmid cloningis also well known. See, e.g., Ausubel, chapter 1.10.11 (supplement 13)and the references therein. See also, Ish-Horowitz and Burke (1981)Nucleic Acids Res. 9:2989-2998; Murray (1983) Phage Lambda and MolecularCloning in Lambda II (Hendrix et al., eds) 395-432 Cold Spring HarborLaboratory, NY; Frischauf et al. (1983) J. Mol. Biol. 170:827-842; and,Dunn and Blattner (1987) Nucleic Acids Res. 15:2677-2698, and thereferences cited therein. Construction of BAC and P1 libraries is wellknown; see, e.g., Ashworth et al. (1995) Anal Biochem 224 (2): 564-571;Wang et al. (1994) Genomics 24(3): 527-534; Kim et al. (1994) Genomics22(2): 336-9; Rouquier et al. (1994) Anal Biochem 217(2): 205-9; Shizuyaet al. (1992) Proc Natl Acad Sci U S A 89(18): 8794-7; Kim et al. (1994)Genomics 22 (2): 336-9; Woo et al. (1994) Nucleic Acids Res 22(23):4922-31; Wang et al. (1995) Plant (3): 525-33; Cai (1995) Genomics 29(2): 413-25; Schmitt et al. (1996) Genomics 1996 33(1): 9-20; Kim et al.(1996) Genomics 34(2): 213-8; Kim et al. (1996) Proc Natl Acad Sci USA(13): 6297-301; Pusch et al. (1996) Gene 183(1-2): 29-33; and, Wang etal. (1996) Genome Res 6(7): 612-9. In general, where the desired goal ofa sequencing project is the sequencing of a genome or expression profileof an organism, a library of the organism's cDNA or genomic DNA is madeaccording to standard procedures described, e.g., in the referencesabove. Individual clones are isolated and sequenced, and overlappingsequence information is ordered to provide the sequence of the organism.See also, Tomb et al. (1997) Nature 539-547 describing the whole genomerandom sequencing and assembly of the complete genomic sequence ofHelicobacter pylori; Fleischmann et al. (1995) Science 269:496-512describing whole genome random sequencing and assembly of the completeHaemophilus influenzae genome; Fraser et al. (1995) Science 270:397-403describing whole genome random sequencing and assembly of the completeMycoplasma genitalium genome and Bult et al. (1996) Science273:1058-1073 describing whole genome random sequencing and assembly ofthe complete Methanococcus jannaschii genome.

[0080] The nucleic acids sequenced by this invention, whether RNA, cDNA,genomic DNA, or a hybrid of the various combinations, are isolated frombiological sources or synthesized in vitro. The nucleic acids of theinvention are present in transformed or transfected whole cells, intransformed or transfected cell lysates, or in a partially purified orsubstantially pure form.

Amplification in Microscale Devices—PCR

[0081] Bench scale in vitro amplification techniques suitable foramplifying sequences to provide a nucleic acid e.g., as a diagnosticindicator for the presence of the sequence, or for subsequent analysis,sequencing or subcloning are known.

[0082] In brief, the most common form of in vitro amplification, i.e.,PCR amplification, generally involves the use of one strand of thetarget nucleic acid sequence as a template for producing a large numberof complements to that sequence. As used herein, the phrase “targetnucleic acid sequence” generally refers to a nucleic acid sequence, orportion of a nucleic acid sequence that is the subject of a particularfluidic operation, e.g., analysis, amplification, identification or thelike. Generally, two primer sequences complementary to different ends ofa segment of the complementary strands of the target sequence hybridizewith their respective strands of the target sequence, and in thepresence of polymerase enzymes and nucleoside triphosphates, the primersare extended along the target sequence through the action of thepolymerase enzyme. The extensions are melted from the target sequence byraising the temperature of the reaction mixture, and the process isrepeated, this time with the additional copies of the target sequencesynthesized in the preceding steps. PCR amplification typically involvesrepeated cycles of denaturation, hybridization and extension reactionsto produce sufficient amounts of the target nucleic acid, all of whichare carried out at different temperatures. Typically, melting of thestrands, or heat denaturation, involves temperatures ranging from about90° C. to 100° C. for times ranging from seconds to minutes. Thetemperature is then cycled down, e.g., to between about 40° C. and 65°C. for annealing, and then cycled up to between about 70° C. and 85° C.for extension of the primers along the target strand.

[0083] Examples of techniques sufficient to direct persons of skillthrough in vitro amplification methods, including the polymerase chainreaction (PCR) the ligase chain reaction (LCR), Qβ-replicaseamplification and other RNA polymerase mediated techniques (e.g., NASBA)are found in Berger, Sambrook, and Ausubel, as well as Mullis et al.,(1987) U.S. Pat. No. 4,683,202; PCR Protocols A Guide to Methods andApplications (Innis et al. eds) Academic Press Inc. San Diego, Calif.(1990) (Innis); Arnheim & Levinson (Oct. 1, 1990) C&EN 36-47; TheJournal of NIH Research (1991) 3, 81-94; (Kwoh et al. (1989) Proc. Natl.Acad. Sci. USA 86, 1173; Guatelli et al. (1990) Proc. Natl. Acad. Sci.USA 87, 1874; Lomell et al. (1989) J. Clin. Chem 35, 1826; Landegren etal., (1988) Science 241, 1077-1080; Van Brunt (1990) Biotechnology 8,291-294; Wu and Wallace, (1989) Gene 4, 560; Barringer et al. (1990)Gene 89, 117, and Sooknanan and Malek (1995) Biotechnology 13: 563-564.Improved methods of cloning in vitro amplified nucleic acids aredescribed in Wallace et al., U.S. Pat. No. 5,426,039. Improved methodsof amplifying large nucleic acids by PCR are summarized in Cheng et al.(1994) Nature 369: 684-685 and the references therein, in which PCRamplicons of up to 40 kb are generated. One of skill will appreciatethat essentially any RNA can be converted into a double stranded DNAsuitable for restriction digestion, PCR expansion and sequencing usingreverse transcriptase and a polymerase. See, Ausbel, Sambrook andBerger, all supra.

[0084] It will be appreciated that these benchtop uses for PCR areadaptable to microfluidic systems. Indeed, PCR amplification isparticularly well suited to use in the apparatus, methods and systems ofthe invention.

[0085] Thermocycling amplification methods, including PCR and LCR, areconveniently performed in microscale devices, making iterative fluidicoperations involving PCR well suited to use in methods and devices ofthe present invention (see also, U.S. Pat. Nos. 5,498,392 and 5,587,128to Willingham et al.). Accordingly, in one preferred embodiment,generation of amplicons such as sequencing templates using PCR, ordirect sequencing of nucleic acids by PCR (e.g., using nucleasedigestion as described supra) is performed with the integrated systemsand devices of the invention.

[0086] Thermocycling in microscale devices is described in co-pendingapplication U.S. Ser. No. 60/056058, attorney docket number017646-003800 entitled “ELECTRICAL CURRENT FOR CONTROLLING FLUIDTEMPERATURES IN MICROCHANNELS” filed Sep. 2, 1997 by Calvin Chow, AnneR. Kopf-Sill and J. Wallace Parce and in Ser. No. 08/977,528, filed Nov.25, 1997. In brief, energy is provided to heat fluids, e.g., samples,analytes, buffers and reagents, in desired locations of the substratesin an efficient manner by application of electric current to fluids inmicrochannels. Thus, the present invention optionally uses power sourcesthat pass electrical current through the fluid in a channel for heatingpurposes, as well as for material transport. In exemplary embodiments,the fluid passes through a channel of a desired cross-section (e.g.,diameter) to enhance thermal transfer of energy from the current to thefluid. The channels can be formed on almost any type of substratematerial such as, for example, amorphous materials (e.g., glass,plastic, silicon), composites, multi-layered materials, combinationsthereof, and the like.

[0087] In general, electric current passing through the fluid in achannel produces heat by dissipating energy through the electricalresistance of the fluid. Power dissipates as the current passes throughthe fluid and goes into the fluid as energy as a function of time toheat the fluid. The following mathematical expression generallydescribes a relationship between power, electrical current, and fluidresistance:

POWER=I ² R

[0088] where

[0089] POWER=power dissipated in fluid;

[0090] I=electric current passing through fluid; and

[0091] R=electric resistance of fluid.

[0092] The above equation provides a relationship between powerdissipated (“POWER”) to current (“I”) and resistance (“R”). In some ofthe embodiments, which are directed toward moving fluid in channels,e.g., to provide mixing, electrophoretic separation, or the like, aportion of the power goes into kinetic energy of moving the fluidthrough the channel. However, it is also possible to use a selectedportion of the power to controllably heat fluid in a channel or selectedchannel regions. A channel region suitable for heating is often narroweror smaller in cross-section than other channel regions in the channelstructure, as a smaller cross-section provides higher resistance in thefluid, which increases the temperature of the fluid as electric currentpasses through. Alternatively, the electric current is increased acrossthe length of the channel by increased voltage, which also increases theamount of power dissipated into the fluid to correspondingly increasefluid temperature.

[0093] To selectively control the temperature of fluid at a region ofthe channel, a power supply applies voltage and/or current in one ofmany ways. For instance, a power supply can apply direct current (i.e.,DC) or alternating current (AC), which passes through the channel andinto a channel region which is smaller in cross-section, thereby heatingfluid in the region. This current is selectively adjusted in magnitudeto complement any voltage or electric field that is applied to movefluid in and out of the region. AC current, voltage, and/or frequencycan be adjusted, for example, to heat the fluid without substantiallymoving the fluid. Alternatively, a power supply can apply a pulse orimpulse of current and/or voltage, which passes through the channel andinto a channel region to heat fluid in the region. This pulse isselectively adjusted to complement any voltage or electric field that isapplied to move fluid in and out of the region. Pulse width, shape,and/or intensity can be adjusted, for example, to heat the fluidsubstantially without moving the fluid or to heat the fluid while movingthe fluid. Still further, the power supply can apply any combination ofDC, AC, and pulse, depending upon the application. In practice, directapplication of electric current to fluids in the microchannels of theinvention results in extremely rapid and easily controlled changes intemperature.

[0094] A controller or computer such as a personal computer monitors thetemperature of the fluid in the region of the channel where the fluid isheated. The controller or computer receives current and voltageinformation from, for example, the power supply and identifies ordetects temperature of fluid in the region of the channel. Dependingupon the desired temperature of fluid in the region, the controller orcomputer adjusts voltage and/or current to meet the desired fluidtemperature. The controller or computer also can be set to be “currentcontrolled” or “voltage controlled” or “power controlled” depending uponthe application.

[0095] The region which is heated can be a “coil” which is optionally ina planar arrangement. Transfer of heat from the coil to a reactionchannel through a substrate material is used to heat the reactionchannel. Alternatively, the coil itself is optionally the reactionchannel.

[0096] A voltage is applied between regions of the coil to directcurrent through the fluid for heating purposes. In particular, a powersupply provides a voltage differential between regions of the coil.Current flows between the regions and traverses a plurality of coils orcoil loops (which can be planar), which are defined by a substrate.Shape and size of the coils can influence an ability of current to heatthe fluid in the coil. As current traverses through the fluid, energy istransferred to the fluid for heating purposes. Cooling coils can also beused. As a cooling coil, a fluid traverses from region to region in thecoil, which can be placed to permit heat transfer through a substratefrom a sample. The cooling fluid can be a variety of substancesincluding liquids and gases. As merely an example, the cooling fluidincludes aqueous solutions, liquid or gaseous nitrogen, and others. Thecooling fluid can be moved between regions using any of the techniquesdescribed herein, and others. Further details are found in Chow et al.,supra.

[0097] The introduction of electrical current into fluid causes heat(Joule heating). In the examples of fluid movement herein where thermaleffects are not desired, the heating effect is minimal because, at thesmall currents employed, heat is rapidly dissipated into the chipitself. By substantially increasing the current across the channel,rapid temperature changes are induced that can be monitored byconductivity. At the same time, the fluid can be kept static in thechannel by using alternating instead of direct current. Becausenanoliter volumes of fluid have tiny thermal mass, transitions betweentemperatures can be extremely short. Oscillations between any twotemperatures above 0° C. and below 100° C. in 100 milliseconds have beenperformed.

[0098] Joule heating in microchannels is an example of how a keycomponent of a conventional genomics methods can be dramaticallyimproved in the formats provided herein. PCR takes hours to performcurrently, primarily because it takes a long time for conventionalheating blocks to oscillate between temperatures. In addition, reagentcost is an obstacle to massive experimentation. Both these parametersare altered by orders of magnitude in the LabChip format. FIG. 1 showsamplification of bacteriophage lambda DNA in a 10 nanoliter volume. Itshould be noted that the optical interrogation volume was 400picoliters. At a template concentration of 10 ng/ml, the signal seenstarting at the 27th cycle came from the amplification of approximately80 target molecules. The transition between 68° C. and 94° C. took placein less than 1 second.

[0099] In one aspect, PCR reaction conditions are controlled as afunction of channel geometry. Microfabrication methods permit themanufacture of channels that have precise variations in cross sectionalarea. Since the channel resistance is inversely proportional to thecross sectional area, the temperature varies with the width and depth ofthe channel for a given flow of current. As fluid moves through astructure of varying cross sectional area, its temperature will change,depending on the dimensions of the channel at any given point. Theamount of time it experiences a given temperature will be determined bythe velocity of the fluid flow, and the length of channel with thosedimensions. This concept is illustrated in FIG. 2. Nucleic acids oftypical lengths have a low diffusion coefficient (about 10⁻⁷ cm/sec²).Thus over the time frame necessary to affect amplification, DNA willonly diffuse a few hundred microns. In a given channel, reactions of afew nanoliters will occupy a few millimeters. Thus in devices ofconvenient length (a few centimeters), many PCR reactions can beperformed concurrently yielding new amplification products every fewseconds per channel. In parallel formats, hundreds of separate reactionscan be performed simultaneously. Because of its simplicity, throughputand convenience, this amplification unit is a preferred feature of manyassays herein.

[0100] In FIG. 2, amplification reactions are performed concurrently inseries using biased alternating current to heat the fluid inside thechannel and move material through it. The time for each step of thereaction is controlled by determining the speed of movement and thelength of channel having particular widths. Flow can be reversed toallow a single small channel region to be used for many separateamplifications.

[0101] As depicted, several samples are run simultaneously in channel210. Sample 215 is in narrow channel region 220; in operation, thisregion is heated to, e.g., 95° C. (hot enough to denature nucleic acidspresent in sample 215, but cool enough that thermostable reagents suchas Taq DNA polymerase are relatively stable due to the relative size ofregion 220 and the applied current. Concurrently, wide channel region230 is heated, e.g., to 60° C. (cool enough for binding of primers insample 225 and initiation of polymerase extension), due to the relativesize of region 230 and the applied current. Concurrently, intermediatechannel region 235 is heated, e.g., to 72° C. (hot enough to preventunwanted non-specific primer-target nucleic acid interactions in sample240 and cool enough to permit continued polymerase extension), due tothe relative size of region 235 and the applied current. This processcan be concurrently carried out with a plurality of additional channelregions such as narrow region 245, wide region 250 and intermediateregion 255, with samples 260, 265 and 270.

[0102] Where possible, direct detection of amplified products can beemployed. For example, differentially labeled competitive probehybridization is used for single nucleotide discrimination.Alternatively, molecular beacons or single nucleotide polymeraseextension can be employed. Homogeneous detection by fluorescencepolarization spectroscopy can also be utilized (fluorescencepolarization has been used to distinguish between labeled smallmolecules free in solution or bound to protein receptors).

[0103] If the analysis requires post-PCR processing, a more complexchannel and control structure is used as in the case where the amplifiedproduct is to be typed at a microsatellite locus. Because singlenucleotide separations take time (approximately 5 minutes today), theoutput of the serial amplification unit is optionally analyzed inparallel separations channels following serial to parallel fluidicmanipulation as described herein.

[0104] Where possible, direct detection of amplified products can beemployed. For example, differentially labeled competitive probehybridization is used for single nucleotide discrimination.Alternatively, molecular beacons or single nucleotide polymeraseextension can be employed. Homogeneous detection by fluorescencepolarization spectroscopy can also be utilized (fluorescencepolarization has been used to distinguish between labeled smallmolecules free in solution or bound to protein receptors).

Amplification in Microscal Devices—Non-thermal Methods

[0105] Another example of a fluidic operation requiring multipleiterative fluid manipulations which was previously impracticable forcost reasons, is non-thermal amplification of nucleic acids. Innon-thermal amplification, strand separation is optionally carried outby chemical means. Thus, by “non-thermal amplification” is meant theamplification of nucleic acids without thermal cycling of the reactionmixture to affect the melting and annealing of the nucleic acid strands.In practice, such methods involve the chemical denaturation of nucleicacid strands, followed by dilution or neutralization of the chemicaldenaturant. For example, in one aspect, strand separation is carried outby raising the pH of the reaction mixture to denature the nucleic acidstrands. The pH is then returned to neutral, for annealing andextension. Other chemical denaturants are equally useful to affectstrand separation. For example, chaotropic agents, e.g., urea,formamide, and the like, are employed in place of base.

[0106] Regardless of the chemical denaturant, however, addition of thesematerials will typically result in the denaturing of the enzymes presentin the reaction mixture, e.g., polymerases, in addition to the nucleicacids, and thereby lead to their inactivation. As such, performance ofthis type of amplification at the bench scale, would require largeamounts of expensive enzymes. Further, the additional volume requiredfor adding these enzymes, as well as diluting or neutralizing thedenaturants, would result in cumbersome manipulations, particularlywhere a large number of cycles is performed.

[0107] In the systems, devices and methods of the present invention,non-thermal amplification can be carried out by introducing a sample ortarget nucleic acid into a reaction chamber, channel or zone of amicrofluidic device. The complementary strands of the target nucleicacid are melted apart by introducing a preselected volume of a chemicaldenaturant, which denatures the complementary strands of the nucleicacid. In particularly preferred aspects, denaturation is accomplished byraising the pH of the reaction mixture to approximately 10-13. This isreadily accomplished by introducing an equal volume of dilute NaOH,e.g., approximately 0.2N NaOH).

[0108] Annealing of the primers to the target strand is carried out byremoving the denaturing effects of the denaturant. For example, in thoseaspects where a dilute base is used to denature the nucleic acid, thebase is optionally neutralized by the addition of a similar volume ofdilute acid, e.g., 0.2N HCl. Where chaotropic agents are used, thedenaturing effect can generally be removed by desalting the reactionmixture or the like. A preselected volume containing an effective amountof polymerase enzyme and primer sequences are then added to the reactionmixture, i.e., sufficient to amplify the target sequence. Becausevolumes of reagents are so small in the devices and methods of theinvention, the polymerase need not be thermally or otherwise stable tothe more extreme conditions of the amplification reaction as in PCR.Specifically, denaturation of the nucleic acids will typically result indenaturation of the polymerase enzyme, as well. However, additionalamounts of enzyme can be added back to the amplification mixture.Because small volumes are used, the costs are maintained relatively low.As a result of this, any number of a variety of common polymeraseenzymes can be used, including E. coli DNA polymerases, e.g., E. coliDNA pol I, Klenow fragment, T7 DNA polymerase or the like. Further, onecould operate the system at an elevated temperature and utilizethermally stable Taq polymerases, Pfu DNA polymerase, Bst and Vent, allof which are commercially available.

[0109] The primers anneal to the target nucleic acid and begin theextension process. Denaturation, annealing and extension steps are thenrepeated the desired number of times to sufficiently amplify the targetnucleic acid. Typically, these cycles are repeated from about 10 toabout 100 times, and preferably between about 10 and 50 times.

[0110] A number of modifications are readily made to this amplificationprocess. For example, one can introduce primer sequences into thereaction mixture at the outset, or along with the polymerase enzymes, asindicated. Similarly, following denaturation, it can be desirable todesalt the amplification reaction mixture, e.g., by passing the mixturethrough a chromatographic matrix incorporated into the device or byseparating the desired elements of the reaction mixture byelectrophoresing the mixture in an appropriate medium. Such desaltingcan be particularly useful where other chemical denaturants are used,e.g., urea, etc. In such cases, the denaturing effects of thesechemicals are typically removed by dilution or removal of the denaturantfrom the amplification reaction mixture, i.e., by desalting.

[0111] An example of a microfluidic device for practicing non-thermalamplification is illustrated in FIG. 3. For ease of discussion, theoperation of this device is described with reference to the use of base(NaOH) mediated denaturation and neutralization with acid (HCl). Asshown, the device 300 is illustrated as being fabricated in a planarsubstrate 301, and including a main channel 302 originating from samplereservoir 304 and terminating in waste reservoir 306. The device alsoincludes a transverse channel 308 which intersects the main channel, andhas at its termini, buffer reservoir 310 and waste reservoir 312. Mainchannel 302 is alternately intersected by NaOH introduction channels(314 a, 314 b and 314 c) fluidly connected to reservoirs which containan appropriate concentration of NaOH (316 a, 316 b and 316 c,respectively) and HCl introduction channels (318 a, 318 b and 318 c)which are fluidly connected to reservoirs (320 a, 320 b and 320 c,respectively) which contain an appropriate concentration of HCl, forneutralizing the base. In the direction of flow along the main channel302, from the sample reservoir 304 to the waste reservoir 306, aftereach intersection of the main channel 302 with the HCl introductionchannels, 318 a, 318 b and 318 c, there is disposed within the mainchannel, a desalting region 322 a, 322 b and 322 c, e.g., a portion ofthe channel that includes an appropriate gel exclusion matrix, nucleicacid binding region, or the like, for separating the salts present inthe sample fluid from the amplified nucleic acid. After the desaltingregions, the main channel is intersected by enzyme/NTP introductionchannels 324 a, 324 b and 324 c, which are fluidly connected toreservoirs (326 a, 326 b and 326 c) which contain effective amounts ofan appropriate DNA polymerase, as well as the four nucleosidetriphosphates or deoxynucleoside triphosphates (NTPs). A detectionwindow 328 is shown across the main channel 302 near the terminus of thechannel into waste reservoir, to detect the product of the overallamplification process. Optional separation regions are also provided inthe terminal portion of the main channel 302 between the last desaltingregion 322 c and the final waste reservoir 306.

[0112] In operation, a sample containing a nucleic acid of interest,e.g., that is sought to be amplified, is introduced along withappropriate primer sequences into main channel 302, e.g., via samplereservoir 304. A stream of sample/primer is transported along mainchannel 302 and out to waste reservoir 312 along transverse channel 308,e.g., by applying appropriate voltages at the various reservoirs, asdescribed herein. A measured slug of sample/primer is then pumped intomain channel 302. Slugs of sample are optionally introduced from anexternal source, e.g., from a sampling system, e.g., as described incommonly assigned copending U.S. patent application Ser. No. 08/671,986filed Jun. 28, 1996, and U.S. patent application Ser. No. 08/760,446,filed Dec. 6, 1996, each of which is incorporated herein by reference inits entirety for all purposes.

[0113] Following introduction into the device, the sample/primer mixtureis then transported up to the intersection of main channel 302 and baseintroduction channel 314 a, whereupon the sample is mixed with a streamof NaOH, that is delivered into main channel 302 from reservoir 316 a,thereby denaturing the nucleic acid of interest. The denaturedsample/primer mixture continues down main channel until it reaches theintersection of the main channel with the HClintroduction channel 318 a,whereupon the denatured sample/primer mixture is mixed with theHCl,thereby neutralizing the mixture and allowing the denatured strandsto re-anneal with the primer sequences.

[0114] Following this annealing step, the annealed mixture is thentransported through a desalting region 322 a, to separate the nucleicacid/primers of interest from salts and low molecular weightcontaminants. The desalted, annealed mixture then continues down themain channel until it reaches the intersection of the main channel 302with enzyme/NTP introduction channel 324 a, whereupon the mixture ismixed with an effective amount of DNA polymerase enzyme in combinationwith effective amounts of the four NTPs used for amplification, andother requisite components for amplification, e.g., Mg++, KCl, etc.,whereupon the enzyme will catalyze extension of the primers along thetemplate nucleic acid of interest.

[0115] This process of denaturing/annealing and extending the nucleicacid of interest is continued along the main channel for the desirednumber of cycles. Although the illustrated device only shows sufficientdenaturant/ neutralizer/enzyme channels for three cycles, this is solelyfor ease of discussion. It will be readily appreciated that the numberof cycles can be readily increased by increasing the number of suchchannels in the device.

[0116] It will be readily apparent that a number of different channelgeometries er effective in producing the non-thermal amplificationdevices and systems of the present invention.

Synthesis and Selection of Primers and Primer Sets—Application toMicrofluidic Sequencing

[0117] Oligonucleotides for use as primers or probes, e.g., insequencing or PCR or non-thermal amplification reactions in microfluidicapparatus are typically synthesized chemically according to the solidphase phosphoramidite triester method described by Beaucage andCaruthers (1981), Tetrahedron Letts., 22(20):1859-1862, e.g., using anautomated synthesizer, as described in Needham-VanDevanter et al. (1984)Nucleic Acids Res., 12:6159-6168. Oligonucleotides can also be custommade and ordered from a variety of commercial sources known to personsof skill. Purification of oligonucleotides, where necessary, istypically performed by either native acrylamide gel electrophoresis orby anion-exchange HPLC as described in Pearson and Regnier (1983) J.Chrom. 255:137-149. The sequence of the synthetic oligonucleotides canbe verified using the chemical degradation method of Maxam and Gilbert(1980) in Grossman and Moldave (eds.) Academic Press, New York, Methodsin Enymology 65:499-560.

[0118] While primers can hybridize to any of a number of sequences,selecting optimal primers is typically done using computer assistedconsideration of available sequences and excluding potential primerswhich do not have desired hybridization characteristics, and/orincluding potential primers which meet selected hybridizationcharacteristics. This is done by determining all possible nucleic acidprimers, or a subset of all possible primers with selected hybridizationproperties (e.g., those with a selected length, G:C ratio, uniqueness inthe given sequence, etc.) based upon the known sequence. The selectionof the hybridization properties of the primer is dependent on thedesired hybridization and discrimination properties of the primer. Ingeneral, the longer the primer, the higher the melting temperature. Inaddition, it is more difficult to generate a set of primers whichincludes all possible oligonucleotides for a given length, as therequired number of primers increases exponentially. For example, allpossible 3-mers requires 4³ primers, all possible 4-mers requires 4⁴primers, all possible 5-mers requires 4⁵ primers, all possible 6-mersrequires 4⁶ primers, etc. Standard sequencing primers are often in therange of 15-20 nucleotides in length, which would require sets of 4¹⁵ to4²⁰ nucleotides, or 1.1×10⁹ to 1.1×10¹² primers.

[0119] While it is possible to make such large sets of primers usingcombinatorial chemical techniques, the associated problems of storingand retrieving billions or even trillions of primers make these primersets less desirable. Instead, smaller sets of primers used in a modularfashion are desirable.

[0120] For example, Ulanovsky and co-workers have described themechanism of the modular primer effect (Beskin et al. (1995) NucleicAcids Research 23(15):2881-2885) in which short primers of 5-6nucleotides can specifically prime a template-dependent polymeraseenzyme when 2-3 contiguously annealing, but unligated, primers are usedin a polymerase dependent reaction such as a sequencing reaction.Polymerase enzymes are preferentially engaged by longer primers, whethermodular or conventional, accounting for the increased specificity ofmodular primers. Because it is possible to synthesize easily allpossible primers with 5-6 nucleotides (i.e., 4⁵ to 4⁶ or 1024 to 4096primers), it is possible to generate and utilize a universal set ofnucleotide primers, thereby eliminating the need to synthesizeparticular primers to extend nucleotide sequencing reactions ofnucleotide templates. In an alternative embodiment, a ligase enzyme isused to ligate primers which hybridize to adjacent portions of atemplate, thereby providing a longer primer.

[0121] A modified version of the use of the modular primer strategy, inwhich small nucleotide primers are specifically elongated for use in PCRto amplify and sequence template nucleic acids has also been described.The procedure is referred to as DNA sequencing using differentialextension with nucleotide subsets (DENS). See, Raja et al. (1997)Nucleic Acids Research 25(4):800-805. Thus, whether standardSanger-style sequencing or direct PCR sequencing using boronatednucleotides and a nuclease (see, Porter et al. 1997, supra.) aredesired, small sets of short primers are sufficient for use insequencing and PCR and are desirable.

[0122] It is expected that one of skill is thoroughly familiar with thetheory and practice of nucleic acid hybridization and primer selection.Gait, ed. Oligonucleotide Synthesis: A Practical Approach, IRL Press,Oxford (1984); W. H. A. Kuijpers Nucleic Acids Research 18(17), 5197(1994); K. L. Dueholm J. Org. Chem. 59, 5767-5773 (1994); S. Agrawal(ed.) Methods in Molecular Biology, volume 20; and Tijssen (1993)Laboratory Techniques in biochemistry and molecularbiology—hybridization with nucleic acid probes, e.g., part I chapter 2“overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, N.Y. provide a basic guide to nucleic acidhybridization. Innis supra provides an overview of primer selection.

[0123] One of skill will recognize that the 3′ end of an amplificationprimer is more important for PCR than the 5′ end. Investigators havereported PCR products where only a few nucleotides at the 3′ end of anamplification primer were complementary to a DNA to be amplified. Inthis regard, nucleotides at the 5′ end of a primer can incorporatestructural features unrelated to the target nucleic acid; for instance,in one embodiment, a sequencing primer hybridization site (or acomplement to such as primer, depending on the application) isincorporated into the amplification primer, where the sequencing primeris derived from a primer used in a standard sequencing kit, such as oneusing a biotinylated or dye-labeled universal M13 or SP6 primer. Thesestructural features are referred to as constant primer regions. Theprimers are typically selected so that there is no complementaritybetween any known target sequence and any constant primer region. One ofskill will appreciate that constant regions in primer sequences areoptional.

[0124] The primers are selected so that no secondary structure formswithin the primer. Self-complementary primers have poor hybridizationproperties, because the complementary portions of the primers selfhybridize (i.e., form hairpin structures). Modular primers are selectedto have minimal cross-hybridization, thereby preventing competitionbetween individual primers and a template nucleic acid and preventingduplex formation of the primers in solution, and possible concatenationof the primers during PCR. If there is more than one constant region inthe primer, the constant regions of the primer are selected so that theydo not self-hybridize or form hairpin structures.

[0125] One of skill will recognize that there are a variety of possibleways of performing the above selection steps, and that variations on thesteps are appropriate. Most typically, selection steps are performedusing simple computer programs to perform the selection as outlinedabove; however, all of the steps are optionally performed manually. Oneavailable computer program for primer selection is the MacVector™program from Kodak. An alternate program is the MFOLD program (GeneticsComputer Group, Madison Wis.) which predicts secondary structure of,e.g., single-stranded nucleic acids. In addition to programs for primerselection, one of skill can easily design simple programs for any or allof the preferred selection steps.

Alternative Sequencing Strategies

[0126] Although the present invention is described for exemplarypurposes as using enzymatic sequencing methods (e.g., using the chaintermination methods of Sanger, or the exonuclease/PCR methods describedabove), it will be appreciated that sequencing by hybridizationprotocols and chemical degradation protocols are also adapted to use inthe present invention.

[0127] In chemical degradation methods, the template is typicallyend-labeled with a radio-active or florescent label and then degradedusing the well-known Maxam-Gilbert method. As applied to the presentinvention, the chemicals used to degrade the nucleic acid aresequentially contacted to the template and the resulting size fragmentsdetected by electrophoresing the fragments through a microchannel asdescribed supra.

[0128] Sequencing by hybridization is generally described, e.g., in U.S.Pat. No. 5,202,231, to Drmanac et al. and, e.g., in Drmanac et al.(1989) Genomics 4:114-128. As adapted to the present invention, amicrofluidic device is provided having a source of labeled primers asdescribed herein, and a source of template to be sequenced. The templateand the labeled primer are hybridized under highly stringent conditions,which permit hybridization to occur only if the primers are perfectlycomplementary to the template. In one embodiment, primers havingcomplementarity to a known region and also having an additional base oradditional bases at the 3′ or 5′ end are separately hybridized to thetemplate; those primers which are perfectly complementary to thetemplate (i.e., where the known and additional bases are perfectlycomplementary to the template) are detected. From the detection of theadditional base or bases, additional primers are selected and theprocess is repeated. Using this strategy, it is possible to sequence theentire template nucleic acid.

[0129] Typically, the sequence is extended by only a single base witheach specific hybridization. This is because, as described supra, it iseasier to make complete sets of small oligonucleotides (e.g., there areonly 4,096 6 nucleotide primers) than it is to make complete sets oflarge oligonucleotides. However, several bases are optionally detectedusing larger primers. One advantage of detecting larger regions ofcomplementarity is that, on average, it is more efficient. It will beappreciated that it is not necessary to test all possible sequences forspecific hybridization if more bases than one adjacent to the knownregions are present in the primers used in the sequencing byhybridization reaction. This is because bases are tested sequentiallyonly until a perfectly complementary sequence to the template is found.Once this primer is determined, additional possible primers for thisregion are not tested; instead, the process is repeated to detect theflanking region. Commonly, the primers have between about 1 and about 4nucleotides which flank known regions of complementarity.

[0130] The detection of hybridization is carried out as described supra.Typically, the template is captured in a region of the microfluidicsubstrate and primers are sequentially contacted to the capturedtemplate under stringent hybridization conditions. After hybridizationand detection of hybridization (e.g., by tracking a fluorescent or aradio-active label on the primer) the primer is washed off of thetemplate (e.g., by varying the salt concentration or heat at the site ofhybridization) and the process is repeated with a second primer.

Integrated Fluidic Operations

[0131] In addition to sequencing applications, the microfluidic devicesand methods herein are useful in performing other operations that relyupon a large number of iterative individual fluid manipulations, e.g.,reagent additions, combinations, apportionings, etc.

Serial Diluter

[0132] The simplest illustration of iterative fluid manipulations in amicrofluidic system is in the serial dilution of fluids. Dilution ofsamples, reagents and the like, is a particularly problematic area inmicrofluidic devices. In particular, when operating at extremely smallvolumes, bleed over effects, diffusion and the like prevent the accuratecontrol and transport of fluids, thereby effectively limiting thedynamic range of dilution available through the device. Accordingly, oneachieves a greater dynamic range of dilution by performing iterativeserial dilutions of a sample fluid. In particular, rather than making asingle 1:100 or 1:1000 dilution, one serially makes 1:10 dilutions toachieve the desired dilution. Because each dilution is relatively minor,fluid control is not as substantial a problem.

[0133] In the devices of the present invention, dilution of a sample istypically carried out in a device that includes a main channelintersected by one or more diluent channels, which are in fluidcommunication with one or more diluent reservoirs, respectively. Asample or reagent is transported, e.g., electroosmotically, along themain channel. Diluent is then transported into the main channel andallowed to mix with the sample, reagent or other fluid for whichdilution is sought. Control of the relative volumes of sample anddiluent is affected by controlling the electrical fields applied to eachof these solutions and which drive their electroosmotic flow within thesystem, as described above. By incorporating multiple diluent channels,one can further increase the range of dilution of which the device iscapable.

Integrated Systems for Assay Normalization

[0134] One similar application of the integrated systems of theinvention is the titration of assay components into the dynamic range ofan assay. For example, an assay can first be performed where one or morecomponents of the assay are not within the range necessary for adequateperformance of the assay, e.g., if the assay is performed using aconcentration which is too high or too low for some components, theassay may not provide quantitative results. This need to titrate assaycomponents into the dynamic range of an assay typically occurs where oneor more component of the assay is present at an unknown activity orconcentration. Ordinarily, the assay must be run at severalconcentrations of components, i.e., the assay is run a first time,components are diluted, the assay is run a second time, etc. until theassay can be performed within the dynamic range of the assay. It will beappreciated that this iterative approach can involve several unknownconcentrations simultaneously, requiring considerable trial and error.

[0135] In the integrated systems of the invention, an assay can beperformed at as many concentrations of components as necessary totitrate the assay components into the dynamic range of the assay, withthe results of each assay being used to optimize additional assaypoints. Similarly, titration curves, which are often the result ofmultiple assay runs with different component concentrations aredetermined by performing repeated assays with different concentrationsof components. Different concentrations of assay components in separateassays can be monitored serially or in parallel.

[0136] The ability to titrate and optimize assays is useful fordiagnostic assays, for determining concentrations or activities ofselected components in a system (proteins, enzymes, nucleic acids, smallmolecules, etc.). Furthermore, the present integrated systems providefor rational selection of assay conditions as data is acquired. Forexample, in one embodiment, a diagnostic assay needs to be performedusing several components which are present at initially unknownconcentrations or activities. A first series of concentration oractivity assays is performed to determine the activity or concentrationof particular components, e.g., enzyme, protein, inhibitor, co-factor,nucleic acid, or the like. After these assays are performed and theconcentrations or activities of some or all of the components for thediagnostic assay are determined, the integrated system selectsappropriate amounts of the assay components, performs any necessarydilutions, combines the assay components and performs the diagnosticassay. Similarly, further data points can be collected by adjusting theconcentrations or amounts of diagnostic assay components and re-runningthe assay. All of the fluid manipulations are performed rapidly and theintegrated system is able to assess and compile the results ofindividual data points or individual assays to select which additionalassays need to be performed for assay verification.

[0137] In its most basic form, assay optimization involves theidentification of all factors affecting a reaction result, followed bythe systematic variation of each of these variables until optimalreaction conditions are identified. This is generally termed an “OFAT”method for “one factor at a time.” Thus, assuming a simple two reagentreaction, one would first identify the factors affecting the outcome,e.g., concentration of reagent A, concentration of reagent B andtemperature. One would then run the assay where one factor was variedwhile the others remained constant. For example, one would perform thesame reaction at numerous different concentrations of reagent A, whilemaintaining the concentration of reagent B and the temperature. Next,reagent B would be varied while reagent A and temperature remainedconstant, and finally, the temperature would be varied.

[0138] Even in this simplest form, the number and complexity ofnecessary reactions is apparent. When one considers that most reactionswill have far more than three variables, and that these variable willnot be independent of each other, the possibility of manually performingthese assays, or even performing them in currently available automatedformats becomes a daunting prospect. For example, while robotic systemsusing microwell plates can perform large numbers of manipulations tooptimize assay parameters, such systems are very expensive. Further, asthese systems are typically limited to the bench scale volumes describedabove, they require large volumes of reagents and large amounts of spacein which to operate.

[0139] The devices, systems and methods of the present invention permitthe optimization of large numbers of different assays, by providing anextremely low volume, automatable and sealed format in which suchoptimization can occur rapidly and automatically. For example, thedevices can run a first fluidic operation by combining a preselectedvolume of a first reactant with a preselected volume of a secondreactant, at a desired or preselected temperature for a desired orpreselected amount of time. The device then repeats the assay, butvaries at least one of the volume of the first or second reactants, thetemperature, or the amount of time allowed for the reaction. This isrepeated until a desired number of varied reactions are performed, i.e.,generating sufficient data to permit an estimation of optimal assayconditions which will produce an optimal result of the reaction, withina desired range of statistical significance. “optimal assay conditions”include those conditions that are required to achieve the desired resultof the reaction. Such desired results can include maximization ofreaction yields, but also includes assay conditions which are optimizedfor sensitivity to one variable, e.g., inhibitor concentration, and thelike.

[0140] An assay optimization using the microfluidic devices and systemsof the invention are illustrated through a competitive binding assay,e.g., antibody-antigen binding. A microfluidic device for performing abinding assay is illustrated in FIG. 4A. As shown, the microfluidicdevice 400 is fabricated into a planar solid substrate 402. The deviceincludes a main channel 404, which includes a separate reaction zone 404a and separation zone 404 b. The device also includes a sample well 406,a first buffer well 408, an antigen well 410, an antibody well 412 and awaste well 414. Second buffer well 416 and waste well 428 are alsoincluded. The main channel 404 is linked to wells 406 through 412 viafluid channels 414-420, respectively. Wells 416 and 428 are linked tothe main separation channel 404 b via channels 422 and 424,respectively. Fluid direction within the device is carried outsubstantially as described herein, e.g., via the concommittentapplication of appropriate electrical voltages at multiple wells. Again,the device includes a detection zone 426 toward one end of the mainchannel, to allow detection of the labeled components as they move alongthe main channel.

[0141] In operation, the antibody panel to be screened against thesample is provided as a mixture or cocktail, and placed in antibody well412. A similar cocktail of the various different, labeled antigens forwhich the sample is being screened is placed in the antigen well 410.Labeling of antigens, or in some cases, antibodies, can be carried outby a variety of well known methods, and can include enzymatic,fluorescent, calorimetric, luminescent or other well known labelingtechniques.

[0142] Initially, an antigen control is run. Specifically, antigen ispumped from well 410 to waste well 428 via channels 418, 404 (throughzones 404 a) and 424. A measured fluid slug or region of labeled antigenis then injected into and pumped along the main channel 404 and throughseparation zone 404 b. The labeled antigens electrophorese into theconstituent antigens, which are flowed past a detector 426. An exampleof data obtainable from the antigen control is shown in FIG. 4B, whereeach of the three peaks represents a different antigen in the antigencocktail. The peak heights for the antigen control are measured forlater use in quantification of the antigen in the sample. From therelative retention times, one can also determine that all of the labeledantigens are present in the cocktail.

[0143] Next, an antigen/antibody complex control is run. In particular,constant streams of antibody and antigen are pumped from theirrespective wells into the main channel 404, and particularly thereaction zone 404 a, and out to waste well 428. A measured slug of themixture is then injected into the separation channel 404 b. Complexationof the antigen with the antibody results in a shift in theelectrophoretic mobility of the labeled complex relative to that of thelabeled antigen alone. FIG. 4C represents data obtainable from theantigen/antibody complex control, where the three peaks detected firstrepresent the uncomplexed labeled antigen, and the last three peaksrepresent the labeled antigen/antibody complex. Of course, in someaspects, electrophoretic mobility is affected in an opposite manner,i.e., resulting in a complex eluting faster than its constituentelements, and both contingencies are envisioned here. Concentrations ofantibody and labeled antigen will also generally be titrated to yieldoptimal responses when contacted with the sample. Methods of titratingthese elements are well known in the art.

[0144] Finally, in a screening run, streams of antibody, antigen andsample are flowed continuously into the reaction channel 404 a and intowaste well 428. A slug of this mixture is then injected into theseparation channel 404 b. Any antigen of interest in the sample willcompete for binding to its counterpart antibody with the correspondinglabeled antigen, resulting in a reduction in the level of labeledcomplex, or an increase in the level of labeled, uncomplexed antigen. Anexample of data obtainable from a test run is shown in FIG. 4D. Asshown, the data would indicate that the sample contains an amount ofantigen AG-1 and AG-3, but little or no AG-2. A quantitativedetermination of the levels of these various antigens within the samplecan be obtained by comparing the peak heights, either labeled,uncomplexed antigen, or labeled complex, from the test run to those ofone or both of the control runs, where the difference (e.g., δ₁ and/orδ₂) is indicative of the amount of antigen in the sample. See, e.g.,Evangelista et al., Am. Clin. Lab. :27-28 (1995).

[0145] Additional wells and channels are optionally provided connectedto different reagent injection channels, e.g., 414-422, to dilute thesevarious elements, in order to optimize the particular assay system.

[0146] Where different antigens, antibodies or complexes thereof, in agiven panel screen lack sufficiently different electrophoreticmobilities, one or more these elements are optionally chemicallymodified, e.g., by the addition of charged groups, to alter theelectrophoretic mobility of that element without substantially affectingthat element's interaction with other elements.

[0147] In performing the above-described assay format, a number ofvariables would be expected to affect the assay performance. A number ofthese variables are set forth in Table 1, with a number of possiblelevels set forth for each variable. TABLE 1 # of Le- Variable velsLevels Sample Conc. 3 low medium high % Ratio of 4 33:33:33 50:25:2525:50:25 25:25:50 [Sample:Ag:Ab] Antibody type 2 Vendor A Vendor B(vendor) Reaction Time 2 0.4 mm/s 0.8 mm/s Reaction Temp. 2 25° C. 37°C. Injected Volume 2 20 pl 50 pl Separation Time 2 0.4 mm/s 0.8 mm/s

[0148] As provided in this ecample, the assay has a total of 7 variabls,each of which has 2, 3 or 4 levels of wariability. In order to perform afull factorial experiment covering these variables, 384 separatereaction runs would be required. Even assuming a ⅛ fractional factorialexperiment, 48 separate runs would be required, which when duplicated,would result in 96 separate runs. When performed at a bench scale, suchan experiment would take hours and would require substantial attentionfrom the investigator to ensure that each assay run is performedcorrectly and accurately. However, in the above described microfluidicformat, each run is automatically performed typically in approximately30 seconds per run. This would permit running all 48 distinct runs, induplicate, in less than one hour (in parallel microfluidic formats, asdiscussed below, the assay could easily be run in a few minutes).Further, the entire experiment is automatically controlled by thecomputer control system of the microfluidic system, as described herein.

[0149] After all of the assays are performed, the results are analyzedand optimal assay conditions are determined. Analysis of these resultsis typically carried out in the control computer system using readilyavailable computer software, designed for experimental optimization,e.g., Design-Ease™ statistical optimization software.

Drug Screening Assays

[0150] In addition to sequencing, the integrated microfluidic system ofthe invention is broadly useful in a variety of screening assays wherethe results of mixing one or more components are to be determined, andparticularly, where the results determined are used to select additionalreagents to be screened.

[0151] As described more fully below, the integrated microfluidic systemof the invention can include a very wide variety of storage elements forstoring reagents to be assessed. These include well plates, matrices,membranes and the like. The reagents are stored in liquids (e.g., in awell on a microtiter plate), or in lyophilized form (e.g., dried on amembrane), and can be transported to an assay component of themicrofluidic device (i.e., a microfluidic substrate having reactionchannels or the like) using conventional robotics, or using anelectropipettor as described below.

[0152] Because of the breadth of the available sample storage formatsfor use with the present invention, virtually any set of reagents can besampled and assayed in an integrated system of the present invention.For example, enzymes and substrates, receptors and ligands, antibodiesand ligands, proteins and inhibitors, cells and growth factors orinhibitors, viruses and virus binding components (antibodies, proteins,chemicals, etc.) immunochemicals and immunoglobulins, nucleic acids andnucleic acid binding chemicals, proteins, or the like, reactantchemicals (acids, bases, organic molecules, hydrocarbons, silicates,etc.) can all be assayed using the integrated systems of the invention.For example, where a molecule which binds a protein is desired,potential binding moieties (chemicals, peptides, nucleic acids, lipids,etc.) are sequentially mixed with the protein in a reaction channel, andbinding is measured (e.g., by change in electrophoretic mobility,quenching of fluorescent protein residues, or the like). Thousands ofcompounds are easily screened using this method, in a short period oftime (e.g., less than an hour).

[0153] An advantage of the integrated nature of the present system isthat it provides for rational selection of structurally or functionallyhomologous compounds or components as the assay progresses. For example,where one compound is found to have binding activity, the selection of asecond compound to be tested can be performed based upon structuralsimilarity to the first active compound. Similarly, where a compound isshown to have activity in a cell (e.g., up-regulation of a gene ofinterest) a second compound affecting the same cellular pathway (e.g.,calcium or inositol phosphate second messenger systems, etc.) can beselected from the group of available compounds for testing. In this way,it is possible to focus screening assays from purely random at theoutset to increasingly focused on likely candidate compounds as theassays progress.

[0154] Further details on drug screening assays adaptable to the presentinvention are found in co-pending application U.S. Ser. No. 08/671,987.

Additional Nucleic Acid Analysis

[0155] Genomic material is subject to a certain amount of variation fromone individual of a particular species to another. For example in amammalian genome of approximately 3 billion base pairs, approximately0.1%, or 3 million base pairs would be expected to vary amongindividuals, and a large number of these variations would be expected tobe linked to or result in potentially important traits.

[0156] A number of methods are currently available for identifying anddistinguishing these variations other than simply sequencing the nucleicacids as described above. For example, Kozal et al., Nature Medicine2(7):753-759 (1996), describes the use of high density oligonucleotideprobe arrays in identifying naturally occurring mutations in HIVinfected patients, which mutations augment resistance to particularantiviral agents, e.g., protease inhibitors.

[0157] Alternative methods for identifying these variations includeactual DNA sequencing discussed above, oligonucleotide ligase assays,including LCR, DNA polymerase based methods, and allele specificamplification methods. Although these methods are generally effective atbenchtop scales when analyzing single or few loci, when comprehensivegenetic information is desired, e.g., requiring analysis of largenumbers of loci, the conditions must be optimized for each locus,requiring the performance of massive numbers of experiments, renderingsuch methods overly expensive, cumbersome and largely impractical.

[0158] In related aspects, the microfluidic devices and systems can bereadily used to perform nucleic acid analysis for identifying andmapping such variations, without the need for amplification orsequencing steps. Briefly, the particular assay system employs ahybridization of a complex nucleic acid sample to groups ofoligonucleotide probes that are complementary to different portions ofthe target sequence and that are immobilized in different regions of areaction channel. Enrichment of a target nucleic acid of interest iscarried out by the iterative hybridization, washing and release of thetarget from these oligonucleotide probes. These probes are optionallycomplementary to different portions of the target or overlappingportions and they are optionally the same or different lengths. Aschematic illustration of a device for such analysis is shown in FIG.5A.

[0159] As shown, the device 500 is again fabricated into a solidsubstrate 502 and includes a main analysis channel 504. The mainanalysis channel includes first, second and third hybridization sites506, 508 and 510, respectively. Each of reservoirs 512-520 are connectedto the main analysis channel by a series of intersecting channels522-536.

[0160] In operation, a sample containing a targeted nucleic acid isplaced in reservoir 516. Where the sample includes double-strandedgenomic DNA, the sample is optionally denatured under basic conditions.This is accomplished, e.g., by delivering a volume of dilute base, e.g.,NaOH, from reservoir 514 via channel 524, to be mixed with the sample atintersection of channels 526 and 524. This intersection optionallycomprises a widened channel or chamber fabricated into the substrate, tofacilitate mixing of the sample and dilute base or to allow for morerefined control of reaction times. The denatured sample is then movedalong channel 528. The dilute base is optionally neutralized bydelivering an equal volume of similarly dilute acid, e.g., HCl,fromreservoir 512, via channel 522, to be mixed with the basic sample at theintersection of channels 522 and 528, which again, can comprise awidened channel or chamber design to facilitate mixing or to allow formore refined control of reaction times. Because samples will typicallyinclude highly complex nucleic acids, this complexity generally preventsthe sample from rapidly re-annealing. The neutralized, denatured sampleis then moved into the main analysis channel 504. Within the mainanalysis channel are hybridization sites 506, 508 and 510, at whichsites are immobilized short, synthetic oligonucleotides that arecomplementary to different portions of a target sequence. Immobilizationof oligonucleotides on solid substrates is optionally carried out by avariety of known methods. For example, often solid supports will includefunctional groups to which oligonucleotides are optionally coupled.Alternatively, substrates are optionally treated to provide such groups,e.g., by silanation of silica substrates.

[0161] These oligonucleotides comprise a set of sequences havinghomology to the target sequence of interest, but not necessarily to eachother, preferably of sequentially increasing lengths along the series ofsites within the main channel 504, such as 10, 15, and 20 nucleotides inlength, at sites 506, 508 and 510 respectively. The lengths of theprobes generally varies depending upon the length and composition of thetarget sequence. The target sequence is preferably at least as long as,if not longer than the longest oligonucleotide. Typically, the probesare arranged in the reaction channel from lowest affinity to highestaffinity in the direction of flow for the gradient of denaturant. Targetsequence that dissociates from the first or weakest affinity probe willthen associate with the next probe in the series, and so on. Statedanother way, the lowest affinity probe will be located in the reactionchannel at a point nearest to the source of denaturant, and willtherefore receive the denaturant gradient first. Probes with strongeraffinity will be located sequentially further from the source ofdenaturant, with the probe having the strongest affinity being furthestfrom the source of denaturant.

[0162] Once directed into the reaction channel 504, the sample ispresented to the first group of probes 506, under conditions suitablefor hybridization to those probes. By “conditions suitable forhybridization” is meant conditions of chemical composition, temperature,and the like, under which the target sequences are capable ofhybridizing to a particular probe sequence. “Stringent hybridization” inthe context of these nucleic acid hybridization experiments are sequencedependent, and are different under different environmental parameters.Generally, highly stringent hybridization conditions are selected to beabout 5°-15° C. lower than the thermal melting point (T_(m)) for thespecific sequence at a defined ionic strength and ph. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget RNA sequence hybridizes to a perfectly matched oligonucleotideprobe. Very stringent conditions are selected to be nearly equal to theT_(m) for a particular probe (e.g., 0°-5° C. below the meltingtemperature). An oligonucleotide “specifically hybridizes” to aparticular target when the probe hybridizes with a least twice thesignal intensity of a control probe. Where the control probe differs byless than 10% (often by only 1 nucleotide) from a test probe, the testprobe is an “allele-specific” probe (to indicate that the test probe canbe used to distinguish between two different alleles of a target whichdiffer by a single nucleotide). See also, Gait, ed. OligonucleotideSynthesis: A Practical Approach, IRL Press, Oxford (1984); W. H. A.Kuijpers Nucleic Acids Research 18(17), 5197 (1994); K. L. Dueholm J.Org. Chem. 59, 5767-5773 (1994); S. Agrawal (ed.) Methods in MolecularBiology, volume 20; and Tijssen (1993) Laboratory Techniques inbiochemistry and molecular biology—hybridization with nucleic acidprobes, e.g., part I chapter 2 “overview of principles of hybridizationand the strategy of nucleic acid probe assays”, Elsevier, N.Y. for abasic guide to nucleic acid hybridization.

[0163] Sequential purification of the target portion of the genome canbe achieved by sequential selective hybridizations to theseoligonucleotides. Thus, for example, when the sequence of interest is 20nucleotides or longer in length, one would expect that a shorteroligonucleotide, such as a 10-mer, will hybridize to many more sites inthe genome than merely the target sequence, just on the statisticalbasis of any particular sequence of 10 nucleotides appearing in thegenome. These sequences will hybridize to the 10-mer oligonucleotide,while non-hybridizing DNA can be washed out of the pool via wastereservoir 538 with diluent in reservoir 518 supplied through channels532 and 536. The reduced pool, which is actually enriched for sequenceshybridizing to the 10-mer, is then subjected to a gradient of denaturantwhich is delivered from reservoir 520. Useful denaturants include thosealready described herein, including, e.g., formamide, and the like. Themaximal concentration of the denaturant is calibrated to maintain amaximal stability of the target sequence/10-mer duplex, therebyeliminating imperfectly hybridized target sequences from the 10-mer orother probes, including double base and single base mismatchedprobe/target hybrids. Determination of optimal levels of denaturant isgenerally carried out experimentally, i.e., by determining optimalhybridization conditions for a given probe sequence.

[0164] Denaturant is transported from reservoir 520 through channels 534and 536, while diluent can be added from reservoir 518 through channel532 to the intersection of channels 532 and 534. Complexity of thesample nucleic acids is substantially reduced by this step. For example,a typical mammalian genome having over 10⁵ base pairs, would be expectedto have approximately 10³ sites capable of hybridizing to a 10-merprobe, effectively allowing a one million-fold reduction in samplecomplexity.

[0165] Following removal of less strongly bound species, the denaturantgradient is restored to a level that causes dissociation of the targetfrom the 10-mer probes, but which permits hybridization to the 15-meroligonucleotide. As a particular sequence of 15 nucleotidesstatistically occurs with less frequency than a 10 nucleotide sequence,again the complexity of the sample DNA will be reduced whennon-hybridized DNA is washed out of the main analysis channel 504. Theseenrichment steps can be performed with oligonucleotides of increasinglength until the desired level of enrichment is achieved. In someembodiments, oligonucleotide probes need not be of increasing length;multiple steps using different oligonucleotide probes will continue toenrich the DNA population for the sequence of interest based onprobability of the oligonucleotide sequence occurring in populations ofdecreasing complexity.

[0166] Preferably, at least one enrichment step is performed beforehybridization to oligonucleotides that “type” the target sequence forthe presence of a particular target sequence or variation.

[0167] Although described in terms of use of chemical denaturants, itwill be readily appreciated that other chemical or non-chemicaltreatments are optionally used to vary the hybridization conditions,including adjusting pH, temperature. Similarly, although varied affinityamong the probes is generally described as being carried out by use ofdifferent length probes, it will also be understood that different probecompositions can also be used to vary affinity of the probe to thetarget. For example, G-C rich probes will hybridize with greateraffinity, i.e., have a higher melting temperature, than A-T rich probes.These chemical properties can be exploited in practicing this aspect ofthe invention.

[0168] Finally, target nucleic acid typed by virtue of its enrichmentand subsequent hybridization to a higher affinity probe, e.g., a 15-meror 20-mer, can be released from the final hybridization site and flowedalong the analysis channel 504. The “typed” target sequence is flowedpast a detection window 540, whereupon it can be detected, i.e., byvirtue of an incorporated labelling group. A variety of direct andindirect labeling and detection methods are well known for nucleicacids, including radiolabeling methods, fluorescent labeling, eitherdirectly or from an intercalating fluorescent dye, chemiluminescentlabeling, calorimetric labeling, labeling with ligands or anti-ligands,e.g., biotin/avidin or streptavidin, and the like.

[0169] In an alternate method, the target sequence can be identified bydetecting the accumulation of the detectable label at the finalhybridization or “typing” site, following the final washing step.

Melting Point Analysis of Nucleic Acids

[0170] In a similar embodiment, the systems, devices and methods of thepresent invention, can be used to detect variations in nucleic acidsequences by determining the strength of the hybridization between thetargeted nucleic acid and probes that are putative perfect complementsto the target. By identifying the difference in stability between theimperfect and perfect hybrids under conditions of increasing hydrogenbond stress, one can identify those nucleic acids that contain avariation.

[0171] In practice, a microfluidic device is configured to accept asample containing an amplified nucleic acid or polynucleotide sequenceof interest, convert it to single-stranded form, facilitatehybridization with a nucleic acid probe, such as an oligonucleotide, andthen subject the hybridization mixture to a chemical or temperaturegradient that distinguishes between perfectly matched targets and thosethat differ by at least one base pair (mismatch). In some embodiments,one or more loci or targeted areas of the sample polynucleotide arefirst amplified by such techniques as PCR or sandwich hybridization. Inother embodiments, unamplified polynucleotide is provided to the deviceand amplified therein, such as in the non-thermal amplificationembodiments described below.

[0172] A schematic illustration of a microfluidic device for carryingout this analysis is shown in FIG. 5B employing the same schematiclayout as the device shown in FIG. 5A. In this aspect, a samplecontaining a nucleic acid is introduced into sample well 516. Thissample is, e.g., introduced into the device, preamplified, or it can betransported to well 516 from another portion of the device where thenucleic acid was amplified, e.g., in integrated operations. Thus,although shown as a well, sample well 516 can be a reservoir or an inletsupplied by an external reservoir or separate reaction chamber. In someembodiments, when the polynucleotide is amplified, one end of theamplified oligonucleotide (e.g., PCR product) is terminated with groupssuch as phosphorothioate bonds that prevent exonucleolytic action byenzymes such as T7 DNA polymerase.

[0173] A preselected amount of amplified target is then fluidicallymoved through channel 526. A preselected amount of exonuclease, e.g., T7DNA polymerase, placed in well 514, is concurrently moved along channel524. Where channel 526 and 524 intersect (intersection 525), the targetand the exonuclease mix in channel 528, and the target is subjected toenzymatic digestion to render the target single-stranded. Alternatively,single stranded target is optionally prepared by asymmetric PCR.

[0174] The resulting single-stranded molecules are then moved alongchannel 528 to the intersection of this channel and probe channel 522.Probe well 512 contains oligonucleotide probes which are putativelycomplementary to a region of the target which contains a potentialvariation. The probe containing solution is delivered along probechannel 522 to the intersection 523 with channel 528, whereupon theprobe solution mixes and hybridizes with the single stranded target. Asabove, a widened channel or chamber is optionally provided at theseintersections to facilitate mixing of the materials.

[0175] Hybridization of the probe results in a perfect hybrid with nomismatches when the sample polynucleotide contains the complementarysequence, i.e., no variation, or in a hybrid with mismatches if thesample polynucleotide differs from the probe, i.e., contains a sequencevariation. The stability of the imperfect hybrid differs from theperfect hybrid under conditions of increasing hydrogen bond stress. Avariety of methods are available for subjecting the hybrids toincreasing hydrogen bond stress, sufficient to distinguish betweenperfectly matched probe/target hybrids and imperfect matches. Forexample, the hybrids are optionally subjected to a temperature gradient,or alternatively, can be subjected to increasing concentrations of achemical denaturant, e.g., formamide, urea, and the like, or increasingpH.

[0176] As shown, the hybridized target/probe mixture is moved throughchannel 530 to the intersection 531 of this channel with denaturantchannel 536. Denaturant, placed in denaturant well 520 is concurrentlydelivered to intersection 531, whereupon it mixes with the target/probehybrid. The denaturant can be diluted with an appropriate diluent buffersupplied from diluent well 518 via channel 532. The differences instability of the hybrids under denaturing conditions can be detected byan integrated separation column such as a capillary channel 528 wheremolecular sieving can be done.

[0177] Following mixing with the denaturant, hybridized andnonhybridized nucleic acids are electrophoretically separated by movingthe mixture along separation channel 504. The separation channel caninclude any of a number of separation matrices, e.g., agarose,polyacrylamide, cellulose, or the like.

[0178] The assay is then repeated several times, varying theconcentration of denaturant with each successive assay. By monitoringthe level of hybrid or single stranded target, one can determine theconcentration of denaturant at which the probe-target hybrid isdenatured. This level is then compared to a standard curve, to determinewhether one or more variations are present.

Microfluidic Detection Apparatus

[0179] The microfluidic apparatus of the invention often, though notnecessarily, comprise a substrate in which fluidic reagents, mixtures ofreagents, reactants, products or the like are mixed and analyzed. A widevariety of suitable substrates for use in the devices of the inventionare described in U.S. Ser. No. 08/761,575, entitled “High ThroughputScreening Assay Systems in Microscale Fluidic Devices” by Parce et al. Amicrofluidic substrate holder is optionally incorporated into thedevices of the invention for holding and/or moving the substrate duringan assay. The substrate holder optionally includes a substrate viewingregion for analysis of reactions carried out on the substrate. Ananalyte detector mounted proximal to the substrate viewing region todetect formation of products and/or passage of reactants along a portionof the substrate is provided. A computer, operably linked to the analytedetector, monitors formation of reactants, separation of sequencingproducts, or the like. An electrokinetic component typically providesfor movement of the fluids on the substrate. Microfluidic devices arealso described in U.S. Ser. No. 08/691,632.

[0180] A principal component of nucleic acid analysis is molecularpartition. Channels in microfluidic substrates can be used for molecularseparations. In addition, the dexterous fluidics in the microfluidicdevices herein produce exquisite control over injection volume—aprincipal parameter determining resolution in molecular partitioning.Aside from biochemistry and analytical capabilities in microdevices,systems that automate access to reagents and specimens are highly usefulfor the integrated systems herein. In high throughput pharmaceuticalscreening a “world-to-chip” interface capable of importing samples fromconventional liquid vessels (such as test tubes or 384-well plates), orfrom solid dots of reagent on substrates is useful. The ability toimport 1000's of different samples with inter-sample intervals as shortas 5 seconds is achieved using the systems herein. A simple system willperform experiments at the rate of 10,000 experiments per channel perday. Simple parallelization of the channels produces a capacity of morethan 1 million such assays per instrument-day.

[0181] Accordingly, in one embodiment, a “sipping” strategy forintroducing solubilized reagents or samples into a microfluidicsubstrate from a standard microplate is used. This is adapted toelements of nucleic acids testing, for example to allow for randomaccess to a library of probes or primers. Although this technologyworks, the advantage of reagent economy that is a hallmark of themicrofluidic technology is somewhat nullified when a chemical librarymust be presented to the system in tens of microliter volumes, e.g., inmicroplates.

[0182] In order to take advantage of the very small quantities ofreagents required by the chip, and to make a system scalable to millionsof experiments, a solid phase reagent interface uniquely suited to highthroughput LabChip processing is desirable. Several new interfaces thatmake use of reagents dried in microarrays on a solid surface aredescribed herein. These configurations are suited to the needs ofdiagnostic products in which elements need to be standardized,convenient, and have acceptable shelf-life. Many robotic systems are nowavailable that can deposit arrays of individual solutions at highdensities (1000 per square centimeter and greater). These are typicallyused as capture elements in heterogeneous phase biochemical assays suchas nucleic acids hybridization. The same approach can be used to depositelements of solution phase reactions (PCR primers, probes, sequencingprimers, etc.). Using these approaches, systems that access solid phasereagents at densities of up to 1000 spots per square centimeter aremade.

[0183] As described above, a preferred integrated method of theinvention incorporates the use of pre-synthesized sets of primers forsequencing and/or PCR, and or reagents to be tested in drug screeningassays. A device of the invention preferably includes a primer storageand/or primer transport mechanism for delivering selected primers to areaction channel in the microfluidic device. Exemplary storagemechanisms optionally include components adapted to holding primers in aliquid or lyophilized form, including containers, containers withseparate compartments, plates with wells (e.g., small microtiter plateswith hundreds or thousands of wells) membranes, matrices, arrays ofpolymers, or the like. Additional embodiments for handling driedreagents on solid substrates are shown below.

[0184] As discussed above, the region for storage of the primers isoptionally located on the substrate of the microfluidic device in fluidconnection to a mixing region or channel on the substrate in which abiochemical reaction (PCR, sequencing or the like) is carried out. In analternative embodiment, the primer storage area is physically separatedfrom the substrate. In this embodiment, the primers can be loaded ontothe substrate, either manually, or using an automated system. Forexample, a Zymate XP (Zymark Corporation; Hopkinton, Mass.) automatedrobot using a Microlab 2200 (Hamilton; Reno, Nev.) pipetting station canbe used to transfer parallel samples to regularly spaced wells in amanner similar to transfer of samples to microtiter plates. If theprimers are stored in lyophilized form (e.g., dried on a substrate), aportion of the lyophilized primer is typically suspended in an aqueoussolution to facilitate transfer to a microfluidic substrate. Anelectropipettor as described above can be used to select and transportsamples to a microfluidic substrate from a well plate, or from anyregion of a microfluidic substrate. Because integration of theelectropipettor with the microfluidic substrates of the invention isrelatively simple, electropipettor embodiments are preferred.

[0185] In preferred embodiments including an electropipettor, a varietyof storage systems for storing reagents, such as primers for delivery tothe microfluidic devices of the invention, are applicable. Compounds areconveniently sampled with the electropipettor from well plates, or fromimmobilized samples stored on a matrix (e.g., a porous, hydrophilic, orhydrophobic matrix), or from dried samples stored on a substrate such asa nitrocellulose, nylon or nytran membrane. In embodiments where thesamples are dried, the samples are solubilized using theelectropipettor, which can be operated to expel a small volume of fluidonto the dried reagent, followed by pipetting the expelled fluidcomprising the reagent into the electropipettor. See also, U.S. Ser. No.08/671,986.

[0186] Accordingly, the present invention provides sampling systemswhich provide the compounds to be sampled in an immobilized format on amembrane matrix or the like, i.e., that the sample material is providedin a fixed position, either by incorporation within a fixed matrix,e.g., a porous matrix, a charged matrix, a hydrophobic or hydrophilicmatrix, or the like, which maintains the sample in a given location.Alternatively, such immobilized samples include samples spotted anddried upon a given sample matrix. In preferred aspects, the compounds tobe screened are provided on a sample matrix in dried form. Typically,such sample matrices will include any of a number of materials that canbe used in the spotting or immobilization of materials, including, e.g.,membranes, such as cellulose, nitrocellulose, PVDF, nylon, polysulfoneand the like. Typically, flexible sample matrices are preferred, topermit folding or rolling of the sample matrices which have largenumbers of different sample compounds immobilized thereon, for easystorage and handling.

[0187] Generally, samples are optionally applied to the sample matrix byany of a number of well known methods. For example, sample libraries arespotted on sheets of a sample matrix using robotic pipetting systemswhich allow for spotting of large numbers of compounds. Alternatively,the sample matrix is treated to provide predefined areas for samplelocalization, e.g., indented wells, or hydrophilic regions surrounded byhydrophobic barriers, or hydrophobic regions surrounded by hydrophilicbarriers (e.g., where samples are originally in a hydrophobic solution),where spotted materials will be retained during the drying process. Suchtreatments then allow the use of more advanced sample applicationmethods, such as those described in U.S. Pat. No. 5,474,796, wherein apiezoelectric pump and nozzle system is used to direct liquid samples toa surface. Generally, however, the methods described in the '796 patentare concerned with the application of liquid samples on a surface forsubsequent reaction with additional liquid samples. However, thesemethods are readily modified to provide dry spotted samples on asubstrate. Similarly, the use of ink-jet printing technology to printbiological and chemical reagents onto substrates is well developed. See,e.g., Wallace (1996) Laboratory Automation News 1(5):6-9 where ink-jetbased fluid microdispensing for biochemical applications is described.

[0188] Similarly, cleavable linkers attaching compounds to an array canbe used to store the compounds in an array, followed by cleavage fromthe array. A variety of cleavable linkers, including acid cleavablelinkers, light or “photo” cleavable linkers and the like are known inthe art. Exemplar arrays are described in Pirrung et al., U.S. Pat. No.5,143,854 (see also, PCT Application No. WO 90/15070), Fodor et al., PCTPublication No. WO 92/10092 Fodor et al. (1991) Science, 251: 767-777;Sheldon et al. (1993) Clinical Chemistry 39(4): 718-719; Kozal et al.(1996) Nature Medicine 2(7): 753-759 and Hubbell U.S. Pat. No.5,571,639. Immobilization of assay components in an array is typicallybe via a cleavable linker group, e.g., a photolabile, acid or baselabile linker group. Accordingly, the assay component is typicallyreleased from the assay e.g., by exposure to a releasing agent such aslight, acid, base or the like prior to flowing the test compound downthe reaction channel. Typically, linking groups are used to attachpolymers or other assay components during the synthesis of the arrays.Thus, preferred linkers operate well under organic and/or aqueousconditions, but cleave readily. under specific cleavage conditions. Thelinker is optionally provided with a spacer having active cleavablesites. In the particular case of oligonucleotides, for example, thespacer is selected from a variety of molecules which can be used inorganic environments associated with synthesis as well as aqueousenvironments, e.g., associated with nucleic acid binding studies.Examples of suitable spacers are polyethyleneglycols, dicarboxylicacids, polyamines and alkylenes, substituted with, for example, methoxyand ethoxy groups. Linking groups which facilitate polymer synthesis onsolid supports and which provide other advantageous properties forbiological assays are known. In some embodiments, the linker providesfor a cleavable function by way of, for example, exposure to an acid orbase. Additionally, the linkers optionally have an active site on oneend opposite the attachment of the linker to a solid substrate in thearray. The active sites are optionally protected during polymersynthesis using protecting groups. Among a wide variety of protectinggroups which are useful are nitroveratryl (NVOC)α-methylnitroveratryl(Menvoc), allyloxycarbonyl (ALLOC), fluorenylmethoxycarbonyl (FMOC),α-methylnitro-piperonyloxycarbonyl (MeNPOC), —NH-FMOC groups, t-butylesters, t-butyl ethers, and the like. Various exemplary protectinggroups are described in, for example, Atherton et al., (1989) SolidPhase Peptide Synthesis, IRL Press, and Greene, et al. (1991) ProtectiveGroups In Organic Chemistry, 2nd Ed., John Wiley & Sons, New York, N.Y.

[0189] Other immobilization or spotting methods are similarly employed.For example, where samples are stable in liquid form, sample matricescan include a porous layer, gel or other polymer material which retain aliquid sample without allowing excess diffusion, evaporation or thelike, but permit withdrawal of at least a portion of the samplematerial, as desired. In order to draw a sample into an electropipettor,the pipettor will free a portion of the sample from the matrix, e.g., bydissolving the matrix, ion exchange, dilution of the sample, and thelike.

[0190] Whether the storage substrate is a filter, membrane, microtiterplate or other material holding reagents of interest, the substrate canconveniently be moved using a mechanical armature. Typically, thespatial location (or “physical address”) of the reagents on thesubstrate are known.. The armature moves the substrate relative to themicrofluidic substrate (and electropipettor, where applicable) so thatthe component for transferring reagent from the substrate to thechannels and wells of a microfluidic substrate (e.g., anelectropipettor) contacts the desired reagent. Alternatively, themicrofluidic substrate or electropipettor can be moved by an armaturerelative to the storage substrate to achieve the same effect. Similarly,both the storage substrate and the microfluidic substrate can be movedby the mechanical armature to achieve the same effect. In anotheraspect, the microfluidic substrate, storage substrate or transferringcomponent (e.g., electropipettor) can be manually manipulated by theoperator.

[0191] A variety of electropipettors, including “resolubilization”pipettors for solubilizing dried reagents for introduction intomicrofluidic apparatus are described in Ser. No. 08/671,986, supra. Inbrief, an electropipettor pipettor having separate channels is fluidlyconnected to an assay portion of the microfluidic device (i.e., amicrofluidic substrate having the reaction and/or analysis and/orseparation channels, wells or the like). In one typical embodiment, theelectropipettor has a tip fluidly connected to a channel underelectroosmotic control. The tip optionally includes features to assistin sample transfer, such as a recessed region to aid in dissolvingsamples. Fluid can be forced into or out of the channel, and thus thetip, depending on the application of current to the channel. Generally,electropipettors utilize electrokinetic or “electroosmotic” materialtransport as described herein, to alternately sample a number of testcompounds, or “subject materials,” and spacer compounds. The pipettorthen typically delivers individual, physically isolated sample or testcompound volumes in subject material regions, in series, into the samplechannel for subsequent manipulation within the device. Individualsamples are typically separated by a spacer region of low ionic strengthspacer fluid. These low ionic strength spacer regions have highervoltage drop over their length than do the higher ionic strength subjectmaterial or test compound regions, thereby driving the electrokineticpumping, and preventing electrophoretic bias. On either side of the testcompound or subject material region, which is typically in higher ionicstrength solution, are fluid regions referred to as first spacer regions(also referred to as high salt regions or “guard bands”), that contactthe interface of the subject material regions. These first spacerregions typically comprise a high ionic strength solution to preventmigration of the sample elements into the lower ionic strength fluidregions, or second spacer region, which would result in electrophoreticbias. The use of such first and second spacer regions is described ingreater detail in U.S. patent application Ser. No. 08/671,986, supra.Spacers are not, however, required, particularly in those embodimentswhere transported components such as primers have the same charge andmass. It will be appreciated that embodiments using identically (ornearly identically) sized primers, such as modular primers, can be usedwithout guard bands.

[0192] In FIG. 6A and FIG. 6B, two solid phase samplers are shown,depicting two approaches for accessing dried reagent arrays bymicrofluidic apparatus. FIG. 6A shows micromachined chip 605 havingthree capillary channels 610, 615, and 620. The channels terminate atone end of chip 605 in sample cup 625. Due to the differences in ionicstrength of the solution in channels 610, 615, and 620, application of apotential from channel 610 to the channel 620, will force fluid intosample cup 625 where it can dissolve dried reagent 630. Subsequentapplication of a potential from right channel 610 to central channel 620will draw solubilized reagent 635 into central channel 620. In FIG. 6B,porous substrate (e.g. microchannel alumina) 640 contains dried reagents645. Application of a sufficient voltage from bottom solvent-supplycapillary 650 to chip capillary 655 attached to a microfluidic element(e.g., a channel on a chip; not shown) causes fluid to pass throughporous substrate 640 and into capillary 655 attached to the microfluidicelement. In passing through substrate 640, the fluid dissolves driedreagent 645 and then carries it into the microfluidic element. In bothsystems, substrate 640 is moved, e.g., by robot to position the samplingcapillary over the appropriate reagent site.

[0193] Alternatively, in embodiments omitting an electropipettor, thechannels are individually fluidly connected to a plurality of separatereservoirs via separate channels. The separate reservoirs each contain aseparate test analyte with additional reservoirs being provided forappropriate spacer compounds. The test compounds and/or spacer compoundsare transported from the various reservoirs into the sample channelsusing appropriate fluid direction schemes. In either case, it generallyis desirable to separate the discrete sample volumes, or test compounds,with appropriate spacer regions.

[0194] One of skill will immediately recognize that any, or all of thecomponents of a microfluidic device of the invention are optionallymanufactured in separable modular units, and assembled to form anapparatus of the invention. See also, U.S. Ser. No. 08/691,632, supra.In particular, a wide variety of substrates having different channels,wells and the like are typically manufactured to fit interchangeablyinto the substrate holder, so that a single apparatus can accommodate,or include, many different substrates adapted to control a particularreaction. Similarly, computers, analyte detectors and substrate holdersare optionally manufactured in a single unit, or in separate moduleswhich are assembled to form an apparatus for manipulating and monitoringa substrate. In particular, a computer does not have to be physicallyassociated with the rest of the apparatus to be “operably linked” to theapparatus. A computer is operably linked when data is delivered fromother components of the apparatus to the computer. One of skill willrecognize that operable linkage can easily be achieved using eitherelectrically conductive cable coupled directly to the computer (e.g.,parallel, serial or modem cables), or using data recorders which storedata to computer readable media (typically magnetic or optical storagemedia such as computer disks and diskettes, CDs, magnetic tapes, butalso optionally including physical media such as punch cards, vinylmedia or the like).

Microfluidic Substrates and Electrokinetic Modulators

[0195] Suitable microfluidic substrate materials are generally selectedbased upon their compatibility with the conditions present in theparticular operation to be performed by the device. Such conditions caninclude extremes of pH, temperature, salt concentration, and applicationof electrical fields. Additionally, substrate materials are alsoselected for their inertness to critical components of an analysis orsynthesis to be carried out by the device.

[0196] Examples of useful substrate materials include, e.g., glass,quartz and silicon as well as polymeric substrates, e.g. plastics. Inthe case of conductive or semi-conductive 'substrates, it isoccasionally desirable to include an insulating layer on the substrate.This is particularly important where the device incorporates electricalelements, e.g., electrical fluid direction systems, sensors and thelike. In the case of polymeric substrates, the substrate materials areoptionally rigid, semi-rigid, or non-rigid, opaque, semi-opaque ortransparent, depending upon the use for which they are intended. Forexample, devices which include an optical, spectrographic, photographicor visual detection element, will generally be fabricated, at least inpart, from transparent materials to allow, or at least, facilitate thatdetection. Alternatively, transparent windows of, e.g., glass or quartz,are optionally incorporated into the device for these types of detectionelements. Additionally, the polymeric materials optionally have linearor branched backbones, and can be crosslinked or non-crosslinked.Examples of particularly preferred polymeric materials include, e.g.,polydimethylsiloxanes (PDMS), polyurethane, polyvinylchloride (PVC)polystyrene, polysulfone, polycarbonate, PMMAs and the like.

[0197] In certain embodiments, the microfluidic substrate includes oneor more microchannels for flowing reactants and products. At least oneof these channels typically has a very small cross sectional dimension,e.g., in the range of from about 0.1 μm to about 500 μm. Preferably thecross-sectional dimensions of the channels is in the range of from about1 to about 200 μm and more preferably in the range of from about 0.1 toabout 100 μm, often in the range of about 1 to 100 μm. In particularlypreferred aspects, each of the channels has at least one cross-sectionaldimension in the range of from about 0.1 μm to about 100 μm. It will beappreciated that in order to maximize the use of space on a substrate,serpentine, saw tooth or other channel geometries, are optionally usedto incorporate longer channels on less substrate area, e.g., tofacilitate separation of reaction products or reactants. Substrates areof essentially any size, with area typical dimensions of about 1 cm² to10 cm².

[0198] In general, the microfluidic devices will include one or morechambers, channels or the like, fluidly connected to allow transport offluid among the chambers and/or channels of these devices. By“microfluidic” is generally meant fluid systems, e.g., channels,chambers and the like, typically fabricated into a solid typicallyplanar substrate, and wherein these fluid elements have at least onecross-sectional dimension in the range of from about 0.1 to about 500μm. Typically, the cross sectional dimensions of the fluid elements willrange from about 1 μm to about 200 μm. The term “channel” is definedabove. A “chamber” will typically, though not necessarily, have agreater volume than a channel, typically resulting from an increasedcross-section having at least one dimension from about 10 to about 500μm, although, as for channels, the range can span, e.g., 0.1 to about500 μm. Although generally described in terms of channels and chambers,it will generally be understood that these structural elements areinterchangeable, and the terms are used primarily for ease ofdiscussion. By “fluidly connected” is meant a junction between tworegions, e.g., chambers, channels, wells etc., through which fluidfreely passes. Such junctions may include ports or channels, which canbe clear, i.e., unobstructed, or can optionally include valves, filters,and the like, provided that fluid freely passes through the junctionwhen desired.

[0199] Manufacturing of these microscale elements into the surface ofthe substrates is generally carried out by any number ofmicrofabrication techniques that are known in the art. For example,lithographic techniques are employed in fabricating, e.g., glass, quartzor silicon substrates, using methods well known in the semiconductormanufacturing industries such as photolithographic etching, plasmaetching or wet chemical etching. See, Sorab K. Ghandi, VLSI Principles:Silicon and Gallium Arsenide, NY, Wiley (see, esp. Chapter 10).Alternatively, micromachining methods such as laser drilling, airabrasion, micromilling and the like are employed. Similarly, forpolymeric substrates, well known manufacturing techniques are used.These techniques include injection molding or stamp molding methodswhere large numbers of substrates are produced using, e.g., rollingstamps to produce large sheets of microscale substrates or polymermicrocasting techniques where the substrate is polymerized within amicromachined mold. Polymeric substrates are further described inProvisional Patent Application Serial No. 60/015,498, filed Apr. 16,1996 (Attorney Docket No. 017646-002600), and Attorney Docket Number17646-002610, filed Apr. 14, 1997.

[0200] In addition to micromachining methods, printing methods are alsoused to fabricate chambers channels and other microfluidic elements on asolid substrate. Such methods are taught in detail in U.S. Ser. No.08/987,803 by Colin Kennedy, Attorney Docket Number 017646-004400, filedDec. 10, 1997 entitled “Fabrication of Microfluidic Circuits by PrintingTechniques.” In brief, printing methods such as ink-jet printing, laserprinting or other printing methods are used to print the outlines of amicrofluidic element on a substrate, and a cover layer is fixed over theprinted outline to provide a closed microfluidic element.

[0201] The substrates will typically include an additional planarelement which overlays the channeled portion of the substrate, enclosingand fluidly sealing the various channels. Attaching the planar coverelement is achieved by a variety of means, including, e.g., thermalbonding, adhesives or, in the case of certain substrates, e.g., glass,or semi-rigid and non-rigid polymeric substrates, a natural adhesionbetween the two components. The planar cover element can additionally beprovided with access ports and/or reservoirs for introducing the variousfluid elements needed for a particular screen, and for introducingelectrodes for electrokinetic movement.

[0202] Typically, an individual microfluidic device will have an overallsize that is fairly small. Generally, the devices will have a square orrectangular shape, but the specific shape of the device can be easilyvaried to accommodate the users needs. While the size of the device isgenerally dictated by the number of operations performed within a singledevice, such devices will typically be from about 1 to about 20 cmacross, and from about 0.01 to about 1.0 cm thick.

Serial to Parallel Conversion

[0203] In performing a large number of parallel fluid manipulations, itis often necessary to allocate a single fluid volume among severalseparate channels or chambers for reaction or analysis. For example, asingle sample volume is introduced into a device along a single channel.To perform a panel of desired screens on the sample, or perform the samescreen multiple times, it is necessary to direct portions of the sampleto separate reaction chambers or channels. Similarly, a series ofdiscrete and different sample volumes is individually directed from thesample introduction channel into multiple separate channels. Thisallocation or direction of a single fluid volume or multiple discretefluid volumes from a serial orientation, i.e., a single channel orchamber, to a parallel orientation, i.e., to multiple separate channelsor chambers, is termed “serial to parallel conversion.” This conversionis particularly applicable to the present invention, in which multipleassays can be run in parallel in a first assay screen, the resultsdetected (in serial or parallel) and a second series of parallel assaysselected for a second screen based upon the results of the first screen.

[0204] As applied to the present invention, methods of performingfluidic operations that include a plurality of parallel fluidmanipulations to provide parallel fluidic analysis of sample materialsis therefore provided, as are related apparatus. In the methods amicrofluidic device is provided. The device has at least a firsttransverse reagent introduction channel fluidly connected to a source ofat least one reagent and a source of at least one sample material. Thetransverse channel is fluidly connected to a plurality of parallelreagent reaction channels. A first reagent or mixture of reagents isselected from the source of at least one reagent, and the first reagentis transported through the reagent introduction channel and a portion ofthe reagent is aliqouted as described into at least one parallel reagentreaction channel (typically into several parallel reaction chambers orchannels). A first sample material is selected from the source of atleast one sample material and the first sample material is aliquotedinto at least a first of the plurality of parallel reagent reactionchannels. At least one additional sample material, or at least oneadditional reagent is selected, and the additional sample material oradditional reagent is aliquoted into at least a second of the pluralityof parallel reagent reaction channels. The first sample material and thefirst reagent are contacted in the first reagent reaction channels,causing a reaction of the first sample material and the first reagent.The at least one additional sample material or at least one additionalreagent is contacted with one or more fluid component such as the firstsample material, the first reagent, at least one additional reagent, atleast one additional sample material, a second additional reagent, asecond additional sample material or the like. The first reactionproduct of the first sample material and the first reagent is detected,as is a second reaction product of at least one additional samplematerial or at least one additional reagent and one or more fluidcomponent (i.e., from two parallel reactions in two or more parallelreaction channels). Based upon the first or second reaction product, asecondary reagent and a secondary sample material are selected and theprocess repeated on these secondary components. It will be appreciatedthat this “parallelization” of multiple assays and selection ofadditional assays based upon the results of a first series of assays candramatically speed selection and performance of related assays, e.g., ina drug screening, assay optimization, diagnostic or nucleic acidsequencing context.

[0205] In one aspect, the method comprises parallel analysis of aplurality of sample materials in the parallel channels, in whichmultiple reagents are mixed in a plurality of the parallel channels withmultiple sample materials to form a multiple of products, and, basedupon detection of the multiple products, selecting the secondary samplematerial and secondary reagent. This multiply parallel format canadditionally speed assay development and data acquisition. In oneaspect, the microfluidic device includes the first transverse reagentintroduction channel and at least a second transverse channel, and aplurality of parallel channels intersecting both of the first and secondtransverse channels. In this format, the step of aliquoting the portionof the reagent into at least one parallel reagent reaction channel isperformed by applying a first voltage across the first transversereagent introduction channel and the second transverse channel to drawthe portion of the reagent into the first transverse reagentintroduction channel, whereby the portion of the reagent is present atintersections of the first channel and each of the plurality of parallelchannels; and, applying a second voltage from the first transversechannel to the second transverse channel, whereby a current in each ofthe parallel channels is equivalent, and whereby the portion of thereagent at the intersections of the first transverse channel and each ofthe plurality of parallel channels is moved in to each of the pluralityof parallel channels.

[0206] In a second serial to parallel conversion aspect, methods ofperforming a plurality of separate assays on a single sample areprovided. In these methods a microfluidic device having at least a firsttransverse channel fluidly connected to at least a source of the sample,a plurality of separate parallel channels fluidly connected to the firsttransverse channel, each of the separate channels having disposedtherein reagents for performing a different diagnostic assay, and afluid direction system for concurrently directing a portion of thesample into each of the plurality of parallel channels is provided. Aportion of the sample is transported into each of the parallel channels,whereby the sample and the reagents disposed in the channel undergo areaction. A result of the reaction of the sample and the reagentsdisposed in the channel, for each of the parallel channels is detected.

[0207] Thus, in certain aspects, the devices and systems of the presentinvention generally include novel substrate channel designs to ensureflow of appropriate amounts of fluids in parallel channels, and therebyfacilitate serial to parallel conversion of fluids in these microfluidicdevices.

[0208] Serial to parallel conversion of fluids within a microfluidicdevice is important for a number of reasons. For example, where one isperforming a number of separate analyses on a single sample, serial toparallel conversion can be used to aliquot the single sample among anumber of separate assay channels in a microfluidic device.Alternatively, a number of physically discrete and different samples,e.g., drug candidates, diagnostic samples, or the like, are seriallyintroduced into a single device and allocated among a number ofdifferent parallel channels subjecting the samples to the same ordifferent analyses.

[0209] Schematic illustrations of serial to parallel conversions areshown in FIG. 7A-7D. For example, in FIG. 7A, a single sample fluidregion (701) is shown being converted to a plurality of separatealiquots of the sample fluid, in a series of parallel channels.Alternatively, as shown in FIG. 7B, separate aliquots of the same samplefluid, provided in a serial orientation in a single channel areallocated to each of several separate parallel channels. In aparticularly useful aspect, as shown in FIG. 7C, a plurality ofdifferent compounds (701, 702, 703 and 704) are serially introduced intoa microfluidic channel (top) and then are each redirected to a separateparallel channel for separate analysis or further manipulation. FIG. 7Dalso illustrates a particularly useful application of serial to parallelconversion where a plurality of different samples (701, 702, 703 and704) are serially introduced into a microfluidic channel, and areallocated and redirected among a number of parallel channels, whereineach parallel channel contains a portion of each of the samples andreflects the serial orientation originally presented (bottom). Thus,serial to parallel conversion is also applicable to performing fluidicoperations which require large numbers of iterative or successive fluidmanipulations, i.e., as in high throughput analysis of samples where aplurality of different samples (e.g., 701, 702, 703 and 704) aresubjected to a plurality of different analyses (e.g., in each separateparallel channel). Specifically, separate channels each perform, inparallel, fluidic operations which separately require iterative and/orsuccessive fluid manipulations.

[0210] While serial to parallel conversion is an important aspect offluid control in microfluidic systems, it does present difficulties froma control aspect. For example, fluid flow in electroosmotic systems iscontrolled by and therefore related to current flow between electrodes.Furthermore, resistance in the fluid channels varies as a function ofpath length and width, and thus, different length channels will havedifferent resistances. If this differential in resistance is notcorrected, it can result in the creation of transverse electrical fieldswhich can inhibit the ability of the devices to direct fluid flow toparticular regions within these devices. Specifically, the current, andthus the fluid flow will follow the path of least resistance, e.g., theshortest path. While this problem of transverse electrical fields isoptionally alleviated through the use of separate electrical systems,i.e., electrodes, at the termini of each and every parallel channel,production of devices incorporating all of these electrodes, and controlsystems for controlling the electrical potential applied at each ofthese electrodes are complex, particularly where one is dealing withhundreds to thousands of parallel channels in a single small scaledevice, e.g., 1-2 cm². Accordingly, the present invention providesmicrofluidic devices for affecting serial to parallel conversion, byensuring that current flow through each of a plurality of parallelchannels is at an appropriate level to ensure a desired flow patternthrough those channels or channel networks. FIGS. 8, 9 and 10 illustratea number of methods and substrate/channel designs for accomplishingthese goals.

[0211] In a first embodiment, FIG. 8 illustrates a substrate 800,employing a channel orientation that is optionally used to accomplishserial to parallel conversion or equal fluid flow in parallel channels.The substrate includes main channel 802, which includes electrodesdisposed in each of ports 804 and 806, at the termini of channel 802. Aseries of parallel channels 808-822 and 830-844 terminate in mainchannel 802. The opposite termini of these parallel channels areconnected to parabolic channels 824 and 846, respectively. Electrodesare disposed in ports 826, 828, 848 and 850, which are included at thetermini of these parabolic channels, respectively.

[0212] In operation, a volume of fluid is transported along main channel802 by applying a potential across electrodes 804 and 806. An equalvoltage is applied across electrodes 826 and 828, and 848 and 850,resulting in a net zero flow through the parallel channels. The sampleis optionally present within main channel 802 as a long slug of a singlesample, or multiple slugs of a single or multiple samples. Once thesample fluid or fluids reach the intersection of the main channel withthe parallel channels, e.g., 830-844, it is then pumped through theparallel channels by applying a potential across electrode sets 826:828and 848:850, which results in a fluid flow from parallel channels808-822, to force the samples into parallel channels 830-844. Thecurrent flow in each of the parallel channels 808-822 and 830-844 ismaintained constant or equivalent, by adjusting the length of theparallel channels, resulting in a parabolic channel structure connectingeach of the parallel channels to its respective electrodes. The voltagedrop within the parabolic channel between the parallel channels ismaintained constant by adjusting the channel width to accommodatevariations in the channel current resulting from the parallel currentpaths created by these parallel channels. For example, channel segment824 a, while longer than channel segment 824 b, has the same resistance,because segment 824 a is appropriately wider. Thus, the parabolic designof channels 824 and 846, in combination with their tapering structures,results in the resistance along all of the parallel channels beingequal, resulting in an equal fluid flow, regardless of the path chosen.Generally, determining the dimensions of channels to ensure that theresistances among the channels are controlled as desired, is optionallycarried out by well known methods, and generally depends upon factorssuch as the make-up of the fluids being moved through the substrates.

[0213] In another example, FIG. 9 illustrates how the principles of thepresent invention can be used in a substrate design that employs fewerelectrodes to affect parallel fluid flow. In particular, fluid flowwithin an array of parallel channels is controlled by a single pair ofelectrodes. As shown, substrate 902 includes a plurality of parallelchannels 904-932. These parallel channels each terminate in transversechannels 934 and 936. Transverse channel 934 has a tapered width, goingfrom its widest at the point where it intersects the nearest parallelchannel 904 to the narrowest at the point it intersects the most distantparallel channel 932. Transverse channel 936, on the other hand, goesfrom its widest at the point it intersects parallel channel 932, to thenarrowest where it intersects parallel channel 902. Electrodes areincluded in the ports 938 and 940 at the wide termini of transversechannels 934 and 936, respectively. The dimensions of these taperedchannels are such that the current flow within each of the parallelchannels is equal, thereby permitting equal flow rates in each channel.As shown, transverse or sample introduction channel 942 is oriented sothat it crosses each parallel channel at the same point relative to oneor the other electrode, to ensure that the potential at theintersections of transverse channel 942 and all of the parallel channels904-932 is the same, again, to prevent the formation of transverseelectrical fields, or “shorting out” the array of channels. This resultsin the sample introduction channel 942 being disposed across theparallel channels at a non-perpendicular angle, as shown.

[0214] In operation, a sample fluid, e.g., disposed in port 944, isflowed through transverse channel 942, and across the intersection ofthe parallel channels 904-932 by applying a potential across ports 944and 946. Once the sample is disposed across the one or more desiredparallel channels, e.g., as dictated by the serial to parallelconversion desired (see, FIGS. 7A-7D), a potential is then appliedacross ports 938 and 940, resulting in an equal fluid flow through eachof the parallel channels and injection of the sample fluid into each ofthe desired parallel channels.

[0215]FIG. 10 illustrates still another embodiment for practicing theprinciples set forth herein. In this embodiment, a substrate includes alarge number of parallel channels. For ease of discussion, thesechannels are referred to herein as parallel channels 1004-1010, althoughit should be understood that preferred aspects will include upwards of20, 50, 100, 500 or more separate parallel channels. The parallelchannels 1004-1010 terminate at one end in transverse channel 1012 andat the other end in transverse channel 1014. Electrodes are providedwithin ports 1016 and 1018, and 1020 and 1022 at the termini of thesetransverse channels. In this embodiment, the problems of varying currentwithin the different parallel channels are addressed by providingtransverse channels 1012 and 1014 with sufficient width that voltagevariation across the length of these transverse channels, and thus, asapplied to each parallel channel, is negligible, or nonexistent.Alternatively, or additionally, a single electrode is optionallydisposed along the length of each of these transverse channels to ensureequal current flow at the transverse channel's intersection with eachparallel channel.

[0216] As shown, however, transverse or sample introduction channel 1024intersects each of the parallel channels, and is controlled byelectrodes disposed within ports 1026 and 1028 at the termini of channel1024. As described for FIG. 9, above, the sample introduction channelintersects each parallel channel at a point where the potential appliedto each channel will be equal. In this aspect, however, the channel isarranged substantially parallel to transverse channels 1012 and 1014, aseach parallel channel is subjected to the same voltages.

[0217] In operation, a sample, e.g., disposed in port 1026, is flowedthrough sample channel 1024, across the intersection of the variousparallel channels 1004-1010, by applying a potential across ports 1026and 1028. Once the sample fluid is in its appropriate location, i.e.,across all or a select number of parallel channels, a potential isapplied across ports 1016:1020 and 1018:1022, injecting a plug of sampleinto the parallel channels.

[0218] The efficacy of these serial to parallel conversions was tested.In brief, a solid slug of fluorescent fluid material, e.g., includingfluorescein, rhodamine or the like, was injected through the diagonaltransverse channel by applying a potential across the transversechannel, e.g., at electrodes 944 and 946, such that the sample fluidspanned several of the parallel channels. By applying a potential acrossthe parallel channels, e.g., at electrodes 938 and 940, that portion ofthe fluid region at the intersections of the transverse channel and eachof the parallel channels was pumped down the parallel channels. Thesample fluid regions in each of the parallel channels was observed toflow at the same rate.

Parallel Fluid Manipulations

[0219] As described, the microfluidic systems of the present inventionare also particularly useful in performing fluidic operations thatrequire a large number of parallel fluid manipulations. Preferredsystems can handle processing of raw sample components through analysisof sample nucleic acids. This includes processing of biological samplessuch as blood such that DNA is available for analysis, providing anautosampling system that can access external reagents or samples andimport them for use with the microchip processing components, andprovide assays on the microfluidic apparatus.

[0220] Two assays in the ultrahigh throughput format are particularlycontemplated: (1) size measurement for microsatellite typing of on-chipamplified DNA, and single nucleotide polymorphism (SNP) genotyping ofon-chip amplified DNA. In a particularly preferred aspect, these assaysare run using parallel microfluidics to maximize sample processingpower.

Sample Diagnostics

[0221] One example of a fluid operation that would benefit from theability to perform rapidly large numbers of parallel manipulations isthe screening of a given sample in a number of separate assays. Forexample a single fluid sample from a patient, e.g., blood, serum, salivaor the like, is screened against a number of separate antibodies orantigens for diagnostic testing. In a microfluidic format, thistypically involves the apportioning of a single larger sample volumeinto numerous separate assay channels or chambers, wherein each separatechamber or channel contains reagents for performing a differentdiagnostic assay. For example, in antibody panel screens, each reactionchamber or channel can contain a different antibody or antigen. Suchassay systems include those described in U.S. patent application Ser.No. 08/671,987, filed Jun. 28, 1996, and previously incorporated hereinby reference.

Genotyping

[0222] Genetic analysis generally involves the correlation of measurablephysical traits (the phenotype) with the inheritance of particularversions of genetic elements (the genotype). Genotyping of nucleic acidsamples from a patient typically involves a two step process. Because ofthe complexity of genomic information, the first step usually involvesan operation for reducing the complexity of the sample, or reducing thenumber of molecules in a mixture to be analyzed, into smaller but usefulportions. Once the complexity of the sample is reduced, the less complexsample is then optionally assayed for a particular genotype, or “typed.”This typing can be repeated upon a number of different segments or“loci” from the overall nucleic acid sample.

[0223] Reduction of sample complexity is typically carried out bybiochemical methods that take a subset of the overall sample andconcentrate it relative to, or purify it away from the remainder of thesample. Examples of these biochemical methods include, e.g., amplifyinga specific subset of sample nucleic acids using preselected primers thatflank the desired segment. Alternatively, the desired segment is pulledfrom the larger sample by hybridization with a predefined probe that iscomplementary to all or a portion of the desired segment.

[0224] Once a nucleic acid sample is pared down to a manageablecomplexity, the sample is typed to identify the presence or absence of aparticular variation. Examples of such variations include simplesequence repeats (“SSR”), single nucleotide polymorphism (“SNP”), andsmall insertions or deletions. In the case of SSRS, typing typicallyinvolves a determination of the size of the sample segment, e.g., usingsize-based electrophoretic methods (gel exclusion), which will indicatethe presence or absence of a larger species corresponding to the samplesegment with or without the additional sequence elements. For SNPs andsmaller insertions or deletions, typing can be carried out by sequencingof the sample segment, to identify the base substitution, addition ordeletion. Such sequencing can be carried out by traditional sequencingmethods or by hybridization of the target sequence to oligonucleotidearrays, e.g., as described in U.S. Pat. No. 5,445,934, which is herebyincorporated herein by reference. Alternatively, the SNP or smallerinsertion or deletion can be identified by nuclease digestion of thesegment followed by size-based separation of the portions of thedigested segment. The pattern of fragments is then correlated with thepresence or absence of a particular marker sequence.

[0225] Typically, methods currently utilized in the art in thesegenotyping experiments analyze each of the various different loci of theoverall sample in a serial format. Specifically, the sample nucleic acidis amplified and characterized at a first locus, then at a second locusand so on. Further, such methods also typically utilize equipment thatis only capable of performing a single component of the overall process,e.g., amplification, electrophoresis, sequencing, etc. As set forthabove, the costs in equipment, time and space for performing thesemethods can be quite high, and increases substantially when a largenumber of samples and/or genetic loci are being screened.

[0226] According to the present invention, several if not all of thecomponents of the overall process are integrated into a singlemicrofluidic device. Further, multiple samples or disparate genetic locifrom a single sample are analyzed within a single device, in a parallelorientation. For example, because of the miniature format of themicrofluidic devices, from about 1 to about 500 different genetic locifrom a single nucleic acid sample can be analyzed in parallel, within asingle device.

[0227] An example of a device for carrying out analysis of multiple locion a single nucleic acid sample is shown in FIG. 11. As shown, thedevice 1100, is fabricated in a solid substrate 1102. The deviceincludes a main sample channel 1114 which is intersected by multipleparallel separation channels 1106-1118. Again, the number of theseseparation channels on a single device can vary depending upon thedesired size of the device. As shown, each of parallel separationchannels 1106-1118 is further intersected by reagent introductionchannels 1120-1132, respectively, and includes reaction chambers1134-1146, respectively. The reagent introduction channels 1120-1132have at their termini, reservoirs 1148:1150, 1152:1154, 1156:1158,1160:1162, 1164:1166, 1168:1170, and 1172:1174, respectively. Separationchannels 1106-1118 have at their termini opposite the sampleintroduction channel 1104, reservoirs 1176-1188 for applying a voltageacross the separation channel.

[0228] In operation, a fluid sample introduced into the sampleintroduction channel 1104 is aliquoted among the separate parallelseparation channels 1106-1118 and delivered to reaction chambers1134-1146, respectively. The sample is then treated according to thedesired protocols by introducing into the reaction chambers reagentsfrom the reservoirs at the termini of the reagent introduction channels.Following amplification, the target sequences are subjected to sizebased separation and analysis by transporting the amplified nucleicacids through the separation channels 1106-1118. Where the amplifiedsequence has a size that is different from the expected size of a“normal” individual it is indicative that sequence includes a sequencevariation, i.e., SSR. Alternatively, the amplified sequence is sequencedby well known sequencing methods. Such sequencing methods are optionallyincorporated into the devices described herein. For example, sequencingcan be carried out by the Sanger method by utilizing four of thereaction chambers for incorporation of each of the four ddNTPs.

[0229] Alternative substrate designs can also be used to accomplish thegoals of the device shown. In particular, as described in reference toserial to parallel conversion, above, a single reagent addition channelcan be provided which intersects all of the parallel separationchannels. Reagents are then serially introduced into this main reagentintroduction channel and delivered to the various separation channelsand reaction chambers, using the serial to parallel conversion aspectsdescribed herein. Similarly, instead of providing a separate wastereservoir for each of the separation channels, a single transversechannel is optionally provided intersecting the separation channels attheir termini opposite the sample introduction channel. This singlechannel can be used to drive fluid flow, e.g., by applying a voltage atthe termini of this transverse channel. By reducing the number of portsat which voltage must be controlled, device design and control aresimplified, also as described herein.

Movement of Materials in Microscale Devices

[0230] As noted, the present invention provides microfluidic systems andmethods of using such systems in the performance of a wide variety offluidic operations and fluid manipulations. Microfluidic devices or“microlaboratory systems,” allow for integration of the elementsrequired for performing these operations or manipulations, automation,and minimal environmental effects on the reaction system, e.g.,evaporation, contamination, human error.

[0231] The phrase “selective direction” or “selective control” generallyrefers to the ability to direct or move a particular fluid volume fromone area in a microfluidic device, e.g., a chamber or channel, toanother area of the microfluidic device. Thus, selective directionincludes the ability to move one of several fluids contained withinseparate regions of a microfluidic device without disturbing the otherfluids, the direction of a portion of a fluid volume, as well as theability to transport or deliver an amount of a particular fluid from afirst chamber to a selected one of several interconnected chambers.

[0232] Selective flowing, movement and direction of fluids within themicroscale fluidic devices is carried out by a variety of methods. Forexample, the devices optionally include integrated microfluidicstructures, such as micropumps and microvalves, or external elements,e.g., pumps and switching valves, for the pumping and direction of thevarious fluids through the device. Examples of microfluidic structuresare described in, e.g., U.S. Pat. Nos. 5,271,724, 5,277,556, 5,171,132,and 5,375,979. See also, Published U.K. Patent Application No. 2 248 891and Published European Patent Application No. 568 902.

[0233] Although microfabricated fluid pumping and valving systems arereadily employed in the devices of the invention, the cost andcomplexity associated with their manufacture and operation can generallyprohibit their use in mass-produced and potentially disposable devicesas are envisioned by the present invention. The devices of the inventionwill typically include an electroosmotic fluid direction system. Suchfluid direction systems combine the elegance of a fluid direction systemdevoid of moving parts, with an ease of manufacturing, fluid control anddisposability. Examples of particularly preferred electroosmotic fluiddirection systems include, e.g., those described in International PatentApplication No. WO 96/04547 to Ramsey et al., as well as U.S. Ser. No.08/761,575 by Parce et al. and U.S. Ser. No. 08/845,754 to Dubrow et al.

[0234] In brief, these fluidic control systems typically includeelectrodes disposed within reservoirs that are placed in fluidconnection with the channels fabricated into the surface of thesubstrate. The materials stored in the reservoirs are transportedthrough the channel system delivering appropriate volumes of the variousmaterials to one or more regions on the substrate in order to carry outa desired screening assay.

[0235] Material transport and direction is accomplished throughelectrokinesis, e.g., electroosmosis or electrophoresis. In brief, whenan appropriate fluid is placed in a channel or other fluid conduithaving functional groups present at the surface, those groups canionize. For example, where the surface of the channel includes hydroxylfunctional groups at the surface, protons can leave the surface of thechannel and enter the fluid. Under such conditions, the surface willpossess a net negative charge, whereas the fluid will possess an excessof protons or positive charge, particularly localized near the interfacebetween the channel surface and the fluid. By applying an electric fieldalong the length of the channel, cations will flow toward the negativeelectrode. Movement of the positively charged species in the fluid pullsthe solvent with them. An electrokinetic device moves components byapplying an electric field to the components, typically in amicrofluidic channel. By applying an electric field along the length ofthe channel, cations will flow toward a negative electrode, while anionswill flow towards a positive electrode. Movement of the charged speciesin the fluid pulls the solvent with the fluid. The steady state velocityof this fluid movement is generally given by the equation:$v = \frac{\varepsilon \quad \xi \quad E}{4\quad \pi \quad \eta}$

[0236] where v is the solvent velocity, ∈ is the dielectric constant ofthe fluid, ξ is the zeta potential of the surface, E is the electricfield strength, and η is the solvent viscosity. The solvent velocity is,therefore, directly proportional to the surface potential.

[0237] To provide appropriate electric fields, the system generallyincludes a voltage controller that is capable of applying selectablevoltage levels, simultaneously, to each of the reservoirs, includingground. Such a voltage controller can be implemented using multiplevoltage dividers and multiple relays to obtain the selectable voltagelevels. Alternatively, multiple, independent voltage sources are used.The voltage controller is electrically connected to each of thereservoirs via an electrode positioned or fabricated within each of theplurality of reservoirs. In one embodiment, multiple electrodes arepositioned to provide for switching of the electric field direction in amicrochannel, thereby causing the analytes to travel a longer distancethan the physical length of the microchannel.

[0238] Substrate materials are also selected to produce channels havinga desired surface charge. In the case of glass substrates, the etchedchannels will possess a net negative charge resulting from the ionizedhydroxyls naturally present at the surface. Alternatively, surfacemodifications are employed to provide an appropriate surface charge,e.g., coatings, derivatization, e.g., silanation, or impregnation of thesurface to provide appropriately charged groups on the surface. Examplesof such treatments are described in, e.g., Provisional PatentApplication Serial No. 60/015,498, filed Apr. 16, 1996 (Attorney DocketNo. 017646-002600). See also, Attorney Docket Number 17646-002610, filedApr. 14, 1997.

[0239] Modulating voltages are then concomitantly applied to the variousreservoirs to affect a desired fluid flow characteristic, e.g.,continuous or discontinuous (e.g., a regularly pulsed field causing theflow to oscillate direction of travel) flow of receptor/enzyme,ligand/substrate toward the waste reservoir with the periodicintroduction of test compounds. Particularly, modulation of the voltagesapplied at the various reservoirs can move and direct fluid flow throughthe interconnected channel structure of the device in a controlledmanner to effect the fluid flow for the desired screening assay andapparatus.

[0240] While a number of devices for carrying out particular methodsaccording to the invention are described in substantial detail herein,it will be recognized that the specific configuration of these deviceswill generally vary depending upon the type of manipulation or reactionto be performed. The small scale, integratability and self- containednature of these devices allows for virtually any reaction orientation tobe realized within the context of the microlaboratory system.

[0241] Because the microfluidic devices of the invention preferablyemploy electroosmotic fluid direction systems, and are substantiallysealed to the outside environment, excepting reagent, buffer or sampleports, they are capable of performing fluidic operations whilemaintaining precise control of the amounts of different fluids to bedelivered to the different regions of the substrate.

[0242] For example, the sealed nature of the devices preventssubstantial evaporation of fluids from the devices. Evaporation, while aproblem at the bench scale, becomes substantially more problematic whenoperating at the microscale, where loss of minute amounts of fluids canhave a dramatic effect on concentrations of the non volatile elements ofthese fluids, particularly where extended reaction times are concerned.Thus, the devices and systems of the invention provide the addedadvantage of performing fluidic operations with a controlled volume. By“controlled volume” is meant that the systems can transport or direct aparticular volume of a particular fluid which is generally within about10% of an expected or desired volume or amount of that fluid, preferablywithin about 5% of an expected or desired volume, and often within about1% of an expected or desired volume.

[0243] The phrase “preselected volume” or simply “selected volume”refers to a volume of fluid that is to be subjected to a particularfluid manipulation. Again, as noted above, in the fluid filled chambers,channels and/or reservoirs of the systems of the invention, thesepreselected volumes are generally transported as slugs of differentfluids within these fluid filled elements. Generally, a preselectedvolume will be within at least about 10% of a desired volume. Thus,where one wishes to transport a preselected volume of 1 μl of aparticular fluid from a first chamber to a second chamber, the fluiddirection systems of the present invention would transport 1 μl±10%. Inpreferred aspects, these systems will maintain a volume within about 5%and often, within about 1%. In addition to reliable volumetric control,the fluid direction systems of the present invention are generallycapable of moving or directing small preselected fluid volumes. Forexample, the fluid direction systems of the present invention aregenerally capable of selectively directing volumes of fluid that areless than about 10 μl, preferably less than about 1 μl, more preferablyless than 0.1 μl and often less than about 10 nl.

[0244] In addition to the volume advantages discussed above, the sealednature and readily automatable fluid direction systems also protectsfluid operation performed in these devices from contaminating influencesfrom the outside environment. Such influences include chemical,biological or microbiological contamination of fluidic operations whichcan affect an outcome of such operations. In addition, suchcontaminating influences can include the occurrence of human error thatis generally associated with manual operations, e.g., measurementerrors, incorrect reagent additions, detection errors and the like.

[0245] High quality data generation is achieved through two basic levelsof control: “hardware-level” control whereby the instruction set forperforming a fluidic operation experiment is coded in fine channels(e.g., 10-100 μm wide, 1-50 μm deep), and “software-level” controlwhereby the movement of fluid and/or materials through the channelnetwork is controlled with exquisite precision by manipulating electricfields introduced into the network through electrodes at channel terminiusing the methods discussed above. Integrated volumetrics capable ofhighly precise, sub-nanoliter measurements and dispensing are a featureof this invention. Electronics that allow simultaneous,millisecond-resolution control over large voltage gradients or currentchanges disposed across the different parts of complex LabChipstructures are performed using the techniques described above. Thispermits fluid or material flow at intersections to be accuratelycontrolled and providing “virtual valves”, structures that meter fluidby electronic control with no moving parts. The electric field controland small conduit dimensions allow experimentation to be performed onsub-nanoliter fluid volumes.

Detectors

[0246] The substrate typically includes a detection window or zone atwhich a signal is monitored. This detection window typically includes atransparent cover allowing visual or optical observation and detectionof the assay results, e.g., observation of a colorometric, fluorometricor radioactive response, or a change in the velocity of colorometric,fluorometric or radioactive component. Detectors often detect a labeledcompound, with typical labels including fluorographic, colorometric andradioactive components. Example detectors include spectrophotometers,photodiodes, microscopes, scintillation counters, cameras, film and thelike, as well as combinations thereof. Examples of suitable detectorsare widely available from a variety of commercial sources known topersons of skill.

[0247] In one aspect, monitoring of the signals at the detection windowis achieved using an optical detection system. For example, fluorescencebased signals are typically monitored using, e.g., in laser activatedfluorescence detection systems which employ a laser light source at anappropriate wavelength for activating the fluorescent indicator withinthe system. Fluorescence is then detected using an appropriate detectorelement, e.g., a photomultiplier tube (PMT). Similarly, for screensemploying colorometric signals, spectrophotometric detection systems areemployed which detect a light source at the sample and provide ameasurement of absorbance or transmissivity of the sample. See also, ThePhotonics Design and Applications Handbook, books 1, 2, 3 and 4,published annually by Laurin Publishing Co., Berkshire Common, P.O. Box1146, Pittsfield, Mass. for common sources for optical components.

[0248] In alternative aspects, the detection system comprisesnon-optical detectors or sensors for detecting a particularcharacteristic of the system disposed within detection window 116. Suchsensors optionally include temperature (useful, e.g., when a reactionproduces or absorbs heat, or when the reaction involves cycles of heatas in PCR or LCR), conductivity, potentiometric (pH, ions), amperometric(for compounds that can be oxidized or reduced, e.g., O₂, H₂O₂, I₂,oxidizable/reducible organic compounds, and the like).

[0249] Alternatively, schemes similar to those employed for theenzymatic system are optionally employed, where there is a signal thatreflects the interaction of the receptor with its ligand. For example,pH indicators which indicate pH effects of receptor-ligand binding canbe incorporated into the device along with the biochemical system, i.e.,in the form of encapsulated cells, whereby slight pH changes resultingfrom binding can be detected. See Weaver, et al., Bio/Technology (1988)6:1084-1089. Additionally, one can monitor activation of enzymesresulting from receptor ligand binding, e.g., activation of kinases, ordetect conformational changes in such enzymes upon activation, e.g.,through incorporation of a fluorophore which is activated or quenched bythe conformational change to the enzyme upon activation.

[0250] One conventional system carries light from a specimen field to acooled charge-coupled device (CCD) camera. A CCD camera includes anarray of picture elements (pixels). The light from the specimen isimaged on the CCD. Particular pixels corresponding to regions of thesubstrate are sampled to obtain light intensity readings for eachposition. Multiple positions are processed in parallel and the timerequired for inquiring as to the intensity of light from each positionis reduced. This approach is particularly well suited to DNA sequencing,because DNA sequencing products are easily labeled using any of avariety of fluorophores known in the art. Many other suitable detectionsystems are known to one of skill.

Computers

[0251] Data obtained (and, optionally, recorded) by the detection deviceis typically processed, e.g., by digitizing the image and storing andanalyzing the image on a computer readable medium. A variety ofcommercially available peripheral equipment and software is availablefor digitizing, storing and analyzing a signal or image. A computer iscommonly used to transform signals from the detection device intosequence information, reaction rates, or the like. PC (Intel x86 orpentium chip-compatible DOS™, OS2™ WINDOWS™ WINDOWS NT™, WINDOWS95™ orWINDOWS97™ based machines), MACINTOSH™, or UNIX™ based (e.g., SUN™ workstation) computers are all commercially common, and known to one ofskill. Software for determining reaction rates or monitoring formationof products, or for translating raw sizing data for sequencing productsinto actual sequence are available, or can easily be constructed by oneof skill using a standard programming language such as Visualbasic,Fortran, Basic, Java, or the like. The software is optionally designedto determine product velocities, concentrations, flux relationships,sequence information and the like as described, supra. Any controller orcomputer optionally includes a monitor which is often a cathode ray tube(“CRT”) display, a flat panel display (e.g., active matrix liquidcrystal display, liquid crystal display), or others. Computer circuitryis often placed in a box which includes numerous integrated circuitchips, such as a microprocessor, memory, interface circuits, and others.The box also optionally includes a hard disk drive, a floppy disk drive,a high capacity removable drive (e.g., ZipDrive™ sold by IomegaCorporation), and other elements. Inputing devices such as a keyboard ormouse optionally provide for input from a person.

[0252] More generally, the microfluidic systems herein typically includecontrol systems for carrying out one or more operations of: controllingfluid movement and direction; monitoring and controlling environmentaleffects on a microfluidic device; and recording and analyzing dataobtained from the microfluidic devices. Typically, such control systemsinclude a programmable computer or processor that is linked, via anappropriate interface, with the other elements of the system. Forexample, the computer or processor will typically interface with: thevoltage controller, to direct the electroosmotic fluid direction system;with a detector disposed adjacent the detection window, to obtain datafrom the device; and with the device itself, to maintain appropriatereaction conditions within the device, e.g., temperature.

[0253] Computerized control of the microfluidic systems allows for therepeated, automatic and accurate performance of the various fluidicoperations performed within a microfluidic device, or within severaldevices, simultaneously. Further, the computer is generally programmableso that a user can modify protocols and/or conditions as desired, aswell as to record, compile and analyze the data from the device, e.g.,statistical analysis. A block diagram of a control system as connectedto a microfluidic device is shown in FIG. 12. In particular, the overallsystem 1200 includes a microfluidic device 1202, a voltage controller1204, a detector 1206, and a computer or processor 1208. The voltagecontroller is connected to electrodes 1210-1216 which are placed inelectrical contact with fluids in the various ports of the microfluidicdevice 1202. The voltage controller is, in turn connected to computer1208. This connection can also include an appropriate AD/DA converter.The computer 1208 is also connected to detector 1206, for instructingoperation of the detector, as well as recording data obtained by thedetector. Detector 1206 is typically disposed adjacent to an appropriatedetection window 1220 within the microfluidic device. In alternateaspects, a detector can be incorporated within the device itself.

Integrated Systems e.g.. for Sequencing, Thermocycling, AssayOptimization and Drug Screening

[0254] The present invention is further illustrated by consideration ofthe accompanying figures.

[0255]FIG. 13 provides an embodiment of the invention having anelectropipettor integrated into a microfluidic substrate having a fluidmixing region, a thermocycler region, a size separation region and adetection region. In operation, reagent storage substrate 1300 havingdried reagent dots, e.g., 1320-1330 is suspended above or belowmicrofluidic substrate 1305 having channels 1345-1355, intersecting atchannel intersections 1360 and 1365 and reagent wells 1370-1390.Channels 1345, 1350 and 1355 are fluidly connected to electropipettor1395 and channel 13100 in electropipettor 1395. As depicted, optionalreagent mixing chamber 13103 provides for mixing of reagents fromsubstrate 1310 prior to entry into channels 1345-1355. In oneembodiment, enzyme for a sequencing reaction (i.e., a polymerase enzyme)is stored in well 1390, while dNTPs are stored in well 1385; thecomponents are mixed e.g., in channel 1365 or channel 1345. This isuseful, e.g., in embodiments where modular primers are used in asequencing reaction and more than one primer is needed for thesequencing reaction. It will be appreciated that chamber 13103 isoptionally omitted, in which case electropipettor channel 13100 andsubstrate channels 1345-1355 are directly connected.

[0256] Electropipettor tip 13105 is fitted to expel fluid onto primerdots 1315-1330 and to then draw the resulting solubilized primer intochannel 13100 for further processing in channels 1345-55, whichoptionally include mixing, heating, or cooling portions. Reactionproducts are separated in channel 1340 having detection zone 13110.Products moving through detection zone 13110 are detected by detector13115 operably coupled to computer 13120. In sequencing embodiments,reagent storage substrate 1310 typically has most or all of the possibleprimers of a given length, e.g., 4,096 6-mer primers, e.g., in 4,096separate dots (optionally more than one primer can exist in a singledot, with the selection of sequencing primers taking all of the primersin each dot into account as compared to the template nucleic acid).Computer 13120 is used to select extension primers from reagent storagesubstrate 1310 according to the selection methods described herein.Sequencing reactions are carried out in channels 1345-1365, optionallyincluding PCR in selected sections of the channels. Sequencing productsare detected by detector 13115, and the detection is converted intosequencing information in computer 13120.

[0257] Although depicted with reagent storage substrate 1310 overmicrofluidic substrate 1335, it will be appreciated that reagent storagesubstrate 1310 can conveniently be either above or below microfluidicsubstrate 1335. In addition, although depicted with dried reagents,reagent storage substrate 1310 can be substituted with a microtiter dishhaving reagents in liquid form, although a microtiter dish will usuallybe located below microfluidic substrate 1335.

[0258]FIG. 14 depicts an alternate embodiment to FIG. 13, in whichelectropipettor 1405 is in the same plane as microfluidic substrate1410. Channels 1415, 14140 and 1430 in substrate 1410 are fluidlyconnected to wells 1435, 1440 and 1445, and are also fluidly connectedto channel 1450 in electropipettor 1405 through optional mixing chamber1407 As depicted, optional reagent mixing chamber 1407 provides formixing of reagents from substrate 1455 prior to entry into channels1415, 1420 and 1430. This is useful, e.g., in embodiments where modularprimers are used in a sequencing reaction and more than one primer isneeded for the sequencing reaction. It will be appreciated that chamber1407 is optionally omitted, in which case electropipettor channel 1450and substrate channels 1415, 1420 and 1430 are directly connected.Reagent storage substrate 1455 having dried reagent dots such as dot1460 is perpendicular (or at an angle) to substrate 1410.Electropipettor 1405 solubilizes dots on reagent storage substrate 1455by expelling liquid from electropipettor tip 1470 onto dots such as dot1460, thereby solubilizing the reagent(s) in dot 1460, and withdrawingthe reagent(s) into electropipettor tip 1470, channel 1450 andsubsequently into substrate 1410. After mixing with additional reagents,e.g. stored in wells 1435, or 1445 and any resulting reaction, reactionproducts are incubated and separated in channel 1420 and detected indetection region 1475 by detector 1480. Waste materials are stored,e.g., in well 1440. Information regarding the detection is digitized andfed into operably linked computer 1485. As discussed above, the computertranslates the information into, e.g., sequence information, drugdiscovery information or the like and directs selection of a secondreagent dot on substrate 1455 (e.g., a second primer) for analysis.

[0259] In the embodiments depicted in FIG. 13 and FIG. 14, computers1320 and 1485 typically store information regarding the location ofreagent dots on reagent storage substrates 1310 and 1455. Typically thiswill be in the form of address information, where the address of eachreagent dot on regent storage substrates 1310 or 1455 is stored forsubsequent selection steps. Either the relevant microfluidic substrate,electropipettor or reagent storage substrate is moved so that theelectropipettor contacts the selected reagent dot (any or all of thecomponents can be moved to cause the electropipettor to contact theproper point on the particular reagent storage substrate. Movement canbe conveniently achieved using a mechanical armature in contact with thecomponent to be moved. Alternatively, the components can be movedmanually.

[0260]FIG. 15 is an alternate preferred embodiment in whichelectropipettor channel 1510 is contiguous with microfluidic channels1520 1530 and 1540 which are connected to wells 1550, 1560 and 1570,respectively. In this embodiment, microfluidic substrate 1580 compriseselectropipettor tip 1590, which includes electropipettor channel 1510.

[0261]FIG. 16 is an additional alternate preferred embodiment in whichelectropipettor capillary 1610 protrudes from microfluidic substrate1620. Capillary 1610 is in fluid communication with microfluidic channel1625, which is in fluid communication with channels 1635, 1640, and 1645and wells 1650-1680.

[0262]FIG. 17 is an additional preferred embodiment similar to thatdepicted in FIG. 15. In operation, electropipettor channel 1710 inelectropipettor tip 1720 is fluidly connected to microfluidic channels1725-1755 and wells 1760-1797 in microfluidic substrate 1799.

[0263] It will be appreciated that the embodiments depicted in FIGS.15-17 can easily by used in an integrated apparatus similar to thatdepicted in FIG. 13 or FIG. 14, i.e., comprising a reagent storagesubstrate, armature for moving the reagent substrate and/or themicrofluidic substrate, a viewing apparatus such as a microscope orphotodiode and a computer for processing data, controlling fluidmovement on the substrate, and controlling movement of electropipettorcomponents relative to the reagent storage substrate.

[0264] Furthermore, it will be appreciated that a variety of reagentstorage substrates are appropriate. For example, FIG. 18 provides apreferred integrated apparatus in which reagents to be selected arestored in liquid form. In operation, liquid reagents are stored inliquid reagent storage tray 1805. The reagents are stored in wells 1810located in reagent storage tray 1805. A variety of reagent storage trayscommercially available are suitable for this purpose, includingmicrotiter dishes, which are available e.g. in a 918-well format.Microfluidic substrate 1815 is located over reagent storage tray 1805.For convenience of manipulation, either microfluidic substrate 1815 ormicrotiter tray 1805 or both can be moved using a robotic armature. Asdepicted, robotic movable armature 1815 is connected to microfluidicsubstrate 1810 and moves the substrate relative to reagent storage tray1805 in response to instructions from computer 1820. Similarly, roboticmovable armature 1825 is attached to and moves reagent storage tray 1805relative to microfluidic substrate 1810 in response to instructions fromcomputer 1820. It will be appreciated that to move microfluidicsubstrate 1810 relative to reagent storage tray 1805, only one movablearmature is need; accordingly, either armature 1815 or armature 1825 isoptionally omitted. Similarly, either armature 1815 or armature 1825 canbe replaced with a movable platform or the like for moving microfluidicsubstrate 1810 relative to reagent storage tray 1805, or vice versa.

[0265] In operation, microfluidic substrate 1810 is sampled byelectropipettor 1830 for sampling reagents from wells 1810.Electropipettor 1830 is fluidly connected to microchannels 1835-1850 andmicrofluidic substrate wells 1860-18100. Reagent mixing, electrophoresisand the like is performed in microchannels 1835-1850. Typically, anelectrokinetic control apparatus such as voltage controller connected toelectrodes located in one or more of microfluidic substrate wells1860-700 controls material transport through microchannels 1835-1850.Detector 18110 detects the results of fluidic mixing assays, such asfluorescent sequencing products, inhibition assays, titrations or thelike. The results detected are digitized and read by computer 1820,which selects additional fluidic reagents for additional assays, basedupon the results detected. Selection of additional reagents causesmovement of movable robotic armature 1825 or 1815, thereby positioningelectropipettor 1830 in well 1810 having the selected reagent.

[0266]FIG. 19 is an outline of the computer processing steps typical indetermining sequence information and in selecting primers useful in themethods and apparatus described herein. Additional processing stepsperformed to run a voltage controller to direct fluid movement in amicrofluidic substrate are optionally performed by the computer.

[0267]FIG. 20 provides an embodiment of the invention directed tosequencing. Template DNAs (e.g., single-stranded cosmid DNA, plasmidDNA, viral DNA or the like) to be sequenced is stored in well 2010. Thetemplate DNAs are conveniently complexed with capture beads. Sequencingreagents (polymerase, dNTPs, ddNTPs or the like) are stored in well2015. Buffers for material transport, and or reagents are stored inwells 2020-2030. Electropipettor channel 2035 is connected to a sourceof all possible 6-mer primers, as described, supra. Template DNA oncapture beads (e.g., posts, magnitic beads, polymer beads or the like)from well 2010 is electrokinetically transported to bead capture area2040. Appropriate primers are selected and transported to bead capture2040 area using electropipettor channel 2035. Polymerase from well 2015is contacted with to template DNA in bead capture area 2040. Extensionof primers on the template with the polymerase results in sequencingproducts. The products are washed from the template using loading bufferfrom well 2020 (the loading buffer optionally comprises a denaturant)and electrophoresed through size separation microchannel 2043. Theproducts separate by size, permitting detection of the products withdetector 2045, which is operatively linked to computer 2050. Afterdetection, products enter waste well 2055. After size detection andanalysis, computer 2050 directs selection of additional primers toextend sequencing of the template DNAs. Once all of the template issequenced by repeated cycles of sequencing, the template and beads arein optional embodiments released from bead capture area 2040 usingbuffer from well 2030 or 2025 and the template DNA beads are transportedto waste well 2060. Additional templates are then loaded into well 2010and the process is repeated with the additional templates.

[0268] The labChip depicted in FIG. 21 was used to perform multipleoperations in a biochemical assay were run on the chip. Thisdemonstrates the ability to integrate functions such as complex (blood)sample preparation, specialized reaction (polymerase chain reaction,PCR), and sophisticated analysis (DNA size separation) in a singleformat.

[0269] In the experiment, LabChip™ 2110 was used to prepare wole blood,load DNA template from whole blood, run the PCR reaction and then sizethe resulting PCR product by gel separation. Channels 2130 and 2140 werefilled with sieving matrix gel 2150. In addition, wells 2160 and 2170 atthe ends of separation channel 2130 were filled with gel. For the firstpart of the experiment, approximately 2000 lymphocytes (white bloodcells) purified from whole blood in a conventional way (centrifugation)were added to 20 μL of PCR reaction mix and placed in sample well 2180of chip 2110. The wells were overlaid with mineral oil and the chip wascycled using a thermocycler. After cycling, the PCR product wasseparated by passage through a second chip through channel 2130. FIG. 22shows the electropherogram for this portion where the amplified peak ofthe HLA locus (about 300 bp) is seen at around 34 seconds at the sametime as the 270-310 bp fragments in the PhiX 174 standard ladder. Forthe second part of the experiment, PCR reaction mix without DNA templatewas placed in well 2180 of a fresh chip and 5% whole blood in which thered blood cells had been lysed was placed in another well. Lymphocytes(white blood cells) were electrophoresed through the channel to the wellcontaining the PCR reaction mixture until 20-100 lymphocytes were in thePCR well. The chip was cycled and DNA separated as for the previouschip. The results are shown in FIG. 23. Amplification was achieved forboth purified and electrophoresed lymphocytes, although the amount ofproduct for purified lymphocytes was larger than for electrophoresedlymphocytes. Sufficient PCR cycles were run to ensure that the reactionhad reached a plateau stage since the number of starting copies wasdifferent. These experiments demonstrate the ability to integrateseveral steps of a complex biochemical assay on a microchip format.

[0270] Modifications can be made to the method and apparatus ashereinbefore described without departing from the spirit or scope of theinvention as claimed, and the invention can be put to a number ofdifferent uses, including:

[0271] The use of an integrated microfluidic system to test the effectof each of a plurality of test compounds in a biochemical system in aniterative process.

[0272] The use of an integrated microfluidic system as hereinbeforedescribed, wherein said biochemical system flows through one of saidchannels substantially continuously, enabling sequential testing of saidplurality of test compounds.

[0273] The use of a microfluidic system as hereinbefore described,wherein the provision of a plurality of reaction channels in said firstsubstrate enables parallel exposure of a plurality of test compounds toat least one biochemical system.

[0274] The use of a microfluidic system as hereinbefore described,wherein each test compound is physically isolated from adjacent testcompounds.

[0275] The use of a substrate carrying intersecting channels inscreening test materials for effect on a biochemical system by flowingsaid test materials and biochemical system together using said channels.

[0276] The use of a substrate as hereinbefore described, wherein atleast one of said channels has at least one cross-sectional dimension ofrange 0.1 to 500 μm.

[0277] The use of an integrated system as described herein for nucleicacid sequencing.

[0278] An assay, kit or system utilizing a use of any one of themicrofluidic components, methods or substrates hereinbefore described.Kits will optionally additionally comprise instructions for performingassays or using the devices herein, packaging materials, one or morecontainers which contain assay, device or system components, or thelike.

[0279] In an additional aspect, the present invention provides kitsembodying the methods and apparatus herein. Kits of the inventionoptionally comprise one or more of the following: (1) an apparatus orapparatus component as described herein; (2) instructions for practicingthe methods described herein, and/or for operating the apparatus orapparatus components herein; (3) one or more assay component; (4) acontainer for holding apparatus or assay components, and, (5) packagingmaterials.

[0280] In a further aspect, the present invention provides for the useof any apparatus, apparatus component or kit herein, for the practice ofany method or assay herein, and/or for the use of any apparatus or kitto practice any assay or method herein.

[0281] While the foregoing invention has been described in some detailfor purposes of clarity and understanding, it will be clear to oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention. For example, all the techniques and apparatusdescribed above can be used in various combinations. All publicationsand patent documents cited in this application are incorporated byreference in their entirety for all purposes to the same extent as ifeach individual publication or patent document were so individuallydenoted.

What is claimed is:
 1. A method of sequencing a nucleic acid comprising:providing a target nucleic acid, a first sequencing primer, apolymerase, dNTPs, and ddNTPs; mixing the target nucleic acid, the firstsequencing primer, the polymerase, the dNTPs, and the ddNTPs in amicrofluidic device under conditions permitting target dependentpolymerization of the dNTPs, thereby providing polymerization products;and, separating the polymerization products by size in the microfluidicdevice to provide a sequence of the target nucleic acid.
 2. The methodof claim 1, wherein a second sequencing primer is selected based uponthe sequence of the target nucleic acid and the second sequencing primeris mixed with the target nucleic acid in a microfluidic device underconditions permitting target dependent elongation of the selected secondsequencing primer thereby providing polymerization products which areseparated by size in the microfluidic device to provide further sequenceof the target nucleic acid.
 3. The method of claim 1, wherein the firstsequencing primer is selected from a large set of sequencing primers byselecting a primer having a sequence complementary to the target nucleicacid.
 4. The method of claim 3, wherein the large set comprises at leastabout 70% of all possible sequencing primers for a given length, whereinthe length is between about 4 and about
 10. 5. The method of claim 3,wherein the large set comprises at least 3,000 different oligonucleotidemembers.
 6. A method of sequencing a target nucleic acid, comprising:(a) providing an integrated microfluidic system comprising amicrofluidic device comprising: at least a first sequencing reactionchannel and at least a first sequencing reagent introduction channel,the sequencing reaction channel and sequencing reagent introductionchannel being in fluid communication; and, a fluidic interface in fluidcommunication with the sequencing reagent introduction channel forsampling a plurality of sequencing reagents or mixtures of sequencingreagents from a plurality of sources of sequencing reagents or mixturesof sequencing reagents and introducing the sequencing reagents ormixtures of sequencing reagents into the sequence reagent introductionchannel from the sources of sequencing reagents or mixtures ofsequencing reagents; selecting a first sequencing primer sequencecomplementary to a first subsequence of a first target nucleic acidsequence; (b) introducing the first sequencing primer and the firsttarget nucleic acid sequence into the sequence reagent introductionchannel; (c) hybridizing the first primer sequence to the firstsubsequence in the first sequencing reaction channel andpolymerase-extending the first primer sequence along the length of thetarget nucleic acid sequence to form a first extension product that iscomplementary to the first subsequence and a second subsequence of thetarget nucleic acid; (d) determining the sequence of the first extensionproduct; (e) based upon the sequence of the first extension product,selecting a second primer sequence complementary to the secondsubsequence of the target nucleic acid sequence; (f) hybridizing thesecond primer sequence to the second subsequence in the first sequencingreaction channel, or, optionally, hybridizing the second primer sequenceto the second subsequence in a second sequencing reaction channel; (g)extending the second primer sequence along the length of the targetnucleic acid sequence to form a second extension product that iscomplementary to the second subsequence and a third subsequence of thetarget nucleic acid sequence; and (h) determining the sequence of thesecond extension product.
 7. The method of claim 1, wherein the secondprimer is selected using a computer.
 8. The method of claim 1, whereinthe microfluidic device comprises a material transport system forcontrollably transporting sequencing reagents through the sequencingreagent introduction channel and sequencing reaction channel.
 9. Amethod of sequencing a nucleic acid comprising: providing a set ofsequencing primers, a target nucleic acid, a polymerase, dNTPs, andddNTPs; selecting a first primer from the set of primers; introducingthe first primer into a microfluidic device; mixing the first primer,the polymerase, the dNTPs, and the ddNTPs in a first zone of themicrofluidic device under conditions permitting target dependentpolymerization of the dNTPs, thereby providing polymerization products;separating polymerization products by size in a second zone of themicrofluidic device to provide at least a first portion of the sequenceof the target nucleic acid; selecting a second primer from the set ofprimers, which primer is complementary to the first portion of thetarget nucleic acid; introducing the second primer into the microfluidicdevice; mixing the second primer, the polymerase, the dNTPs, and theddNTPs in a third zone of the microfluidic device under conditionspermitting target dependent polymerization of the dNTPs, therebyproviding polymerization products; separating polymerization products bysize in a fourth zone of the microfluidic device to provide at least asecond portion of the sequence of the target nucleic acid.
 10. Themethod of claim 9, wherein the first and third zone of the microfluidicdevice are the same and wherein the second and fourth zone of themicrofluidic device are the same.
 11. The method of claim 9, wherein theset of primers is located on the microfluidic apparatus.
 12. The methodof claim 9, wherein mixing the second primer, polymerase, dNTPs, ddNTPsin a microfluidic device under conditions permitting polymerization andseparating polymerization products by size to provide at least a portionof the sequence of the target nucleic acid is performed in less than 15minutes.
 13. The method of claim 9, further comprising selecting a thirdprimer from the set of primers, which third primer is complementary tothe second portion of the target nucleic acid; mixing the third primer,the polymerase, the dNTPs, and the ddNTPs in a microfluidic device underconditions permitting target dependent polymerization of the dNTPs,thereby providing polymerization products; separating polymerizationproducts by size to provide at least a third portion of the sequence ofthe target nucleic acid.
 14. A method of determining a sequence ofnucleotides in a nucleic acid target sequence, comprising: providing amicrofluidic device having a body structure, at least a first analysischannel, and at least a first probe introduction channel disposedtherein, the analysis channel being in fluid communication with a sourceof the target nucleic sequence, and the probe introduction channelintersecting the analysis channel and being in fluid communication witha plurality of sources of extension probes; flowing the target nucleicacid in the analysis channel, wherein a first subsequence of nucleotidesin the target nucleic acid sequence is known; separately injecting eachof a plurality of extension probes into the analysis channel, whereuponthe extension probes contact the target nucleic acid sequence, each ofthe plurality of extension probes having a first sequence portion thatis perfectly complementary to at least a portion of the firstsubsequence, and an extension portion that corresponds to a portion ofthe target nucleic acid sequence adjacent to the target subsequence, theextension portion having a length n, and comprising all possiblenucleotide sequences of length n, wherein n is between 1 and 4inclusive; and, identifying a sequence of nucleotides in the targetnucleic acid adjacent the target subsequence, based upon which of theplurality of extension probes perfectly hybridizes with the targetnucleic acid sequence.
 15. The method of claim 13, wherein n is selectedfrom the group of 1, 2, 3 and
 4. 16. A sequencing apparatus comprising:a body having top portion, a bottom portion and an interior portion; theinterior portion comprising at least two intersecting channels, whereinat least one of the two intersecting channels has at least one crosssectional dimension between about 0.1 μm and 500 μm; an electrokineticfluid direction system for moving a sequencing reagent through at leastone of the two intersecting channels; a source of sequencing primers; anelectropipettor for introducing sequencing primers from the source ofsequencing primers to the at least two intersecting channels; a mixingzone fluidly connected to the at least two intersecting channels formixing the sequencing reagents; a size separation zone fluidly connectedto the mixing zone for separating sequencing products by size, therebyproviding the sequence of a target nucleic acid.
 17. The apparatus ofclaim 16, further comprising a sequence detector for reading thesequence of the target nucleic acid.
 18. The apparatus of claim 16,wherein the source of sequencing primers comprises a set of about 4,096primers.
 19. The apparatus of claim 18, wherein the primers are a set ofapproximately all possible 6 mers.
 20. A system for determining asequence of nucleotides in a target nucleic acid sequence, comprising: amicrofluidic device comprising a body structure, the body structurehaving at least a first analysis channel, and at least a first probeintroduction channel disposed therein, the analysis channel intersectingand being in fluid communication with the probe introduction channel; asource of the target nucleic acid sequence in fluid communication withthe analysis channel; a plurality of separate sources of oligonucleotideprobes in fluid communication with the probe introduction channel, eachof the plurality of separate sources containing an oligonucleotide probehaving a different nucleotide sequence of length n; a sampling systemfor separately transporting a volume of each of the oligonucleotideprobes from the sources of oligonucleotide probes to the probeintroduction channel and injecting each of the oligonucleotide probesinto the analysis channel to contact the target nucleic acid sequence; adetection system for identifying whether each oligonucleotide probehybridizes with the target nucleic acid sequence.
 21. The system ofclaim 20, wherein the plurality of separate sources includes allpossible oligonucleotide sequences of length n.
 22. A method ofdetecting a target nucleic acid sequence in a mixture of nucleic acidsequences comprising: providing a microfluidic device which comprises areaction channel fluidly connected to a source of a chemical denaturant,and wherein the reaction channel has at least first, second and thirdgroups of probes immobilized in first, second and third differentregions of the channel, respectively, the first, second and third probeseach having a different affinity for the target nucleic acid sequence;delivering the sample to the reaction channel under conditions suitablefor hybridization of the target nucleic acid to the firstoligonucleotide probe; transporting sufficient denaturant from thesource of denaturant to the reaction channel whereby the target nucleicacid sequence dissociates from the first probe, but is still capable ofhybridizing to the second probe; transporting sufficient denaturant fromthe source of denaturant to the reaction channel whereby the targetnucleic acid sequence dissociates from the second probe, but is stillcapable of hybridizing to the third probe; transporting sufficientdenaturant from the source of denaturant to the reaction channel wherebythe target nucleic acid sequence dissociates from the third probe; anddetecting the target nucleic acid sequence dissociated from the thirdprobe.
 23. The method of claim 21, wherein the first probe is shorterthan the second probe which is shorter than the third probe, and each ofthe probes is complementary to a different portion of the target nucleicacid sequence.
 24. The method of claim 21, wherein the first region isat a position in the channel nearer the source of chemical denaturantthan the second region, and the second region is nearer the source ofchemical denaturant than the third position.
 25. The method of claim 21,wherein the target nucleic acid incorporates a detectable label and thedetecting step comprises detecting the label.
 26. A method ofdetermining the presence or absence of a sequence variation in a targetnucleic acid sequence, comprising: providing a microfluidic device whichincludes at least a first reaction channel, a source of a target nucleicacid fluidly connected to the reaction channel, a source ofoligonucleotide probes which is complementary to the target nucleic acidsequence fluidly connected to the reaction channel, and a fluiddirection system for transporting the target nucleic acid sequence andthe oligonucleotide probe into the reaction channel; transporting avolume of the target nucleic acid sequence and a volume of theoligonucleotide probes into the reaction channel under conditionssuitable for hybridization of the target nucleic acid sequence to theoligonucleotide probes wherein the target nucleic acid and theoligonucleotide probes have up to two mismatched bases; detecting afirst level of hybridization between the target nucleic acid and theoligonucleotide probes; transporting a volume of the target nucleic acidsequence and the oligonucleotide probes under conditions suitable tohybridization of perfectly matched target nucleic acid sequences andoligonucleotide probes but not suitable for hybridization of targetnucleic acid sequences and oligonucleotide probes that have at least onemismatched base; and, detecting a second level of hybridization of theperfectly matched target nucleic acid sequence and the oligonucleotideprobes, a decrease in the second level of hybridization over the firstlevel of hybridization being indicative of the presence of a sequencevariation in the target nucleic acid sequence.
 27. The method of claim26, wherein the conditions suitable for hybridization of perfectlymatched target nucleic acid sequences and oligonucleotide probes but notsuitable for hybridization of target nucleic acid sequences andoligonucleotide probes that have at least one mismatched base, comprisesmaintaining the reaction channel at a temperature at which onlyperfectly matched target nucleic acid sequences and probes willhybridize.
 28. An integrated method of performing a fluidic analysis ofsample materials, comprising: (a) providing an integrated microfluidicsystem comprising a microfluidic device comprising: at least a firstreaction chamber or channel, and at least a first reagent introductionchannel, the first reaction chamber or channel and reagent introductionchannel being in fluid communication; a material transport system forcontrollably transporting a material through the reagent introductionchannel and reaction chamber or channel; a fluidic interface in fluidcommunication with the reagent introduction channel for sampling aplurality of reagents or mixtures of reagents from a plurality ofsources of reagents or mixtures of reagents and introducing the reagentsor mixtures of reagents into the reagent introduction channel; (b)selecting a first reagent from the plurality of sources of reagent ormixtures of reagents; (c) introducing a first sample material and thefirst reagent or mixture of reagents into the first reaction chamber orchannel whereupon the first sample material and the first reagent ormixture of reagents react; (d) analyzing a reaction product of the firstsample material and the first reagent or mixture of reagents; (e) basedupon the reaction product of step (d), selecting a second reagent ormixture of reagents and a second sample material; (f) introducing thesecond reagent or mixture of reagents into the first reaction chamber orchannel, or, optionally, into a second reaction chamber or channel inthe microfluidic device, whereupon the second sample material and thesecond reagent or mixture of reagents react; and (g) analyzing a secondreaction product of the second sample material and the second reagent ormixture of reagents, thereby providing a fluidic analysis of the firstand second sample materials.
 29. The method of claim 28, wherein thefirst reaction chamber or channel is a reaction channel.
 30. The methodof claim 28, wherein the second reaction chamber or channel is areaction channel.
 31. The method of claim 28, wherein the first andsecond sample materials comprise the same sample constituents.
 32. Themethod of claim 28, wherein the first mixture of reagents and the secondmixture of reagents comprise the same reagent components.
 33. The methodof claim 28, wherein: the first material comprises a first DNA template;the second material comprises a second DNA template; the first mixtureof reagents comprises a first set of DNA sequencing reagents comprisinga first nucleic acid sequencing primer; the second mixture of reagentscomprises a second set of DNA sequencing reagents comprising a secondnucleic acid sequencing primer; the first reaction product comprises theproducts of DNA sequencing; and wherein analyzing the product in step(d) comprises separating the DNA sequencing products by size anddetecting the size separated DNA sequencing products, thereby providingsequence information for the first DNA template; and wherein the methodcomprises the step of selecting a second sequencing primer for inclusionin the second mixture of reagents.
 34. The method of claim 33, whereinthe first or second reaction mixtures comprise a thermostable polymeraseand the method further comprises heating the sample materials and thefirst or second reaction mixture.
 35. The method of claim 33, whereinthe second sequencing primer is selected using a computer.
 36. Themethod of claim 28, wherein first and second materials are bothintroduced into the first reaction chamber or channel.
 37. The method ofclaim 28, wherein the method is substantially free from contamination.38. The method of claim 28, wherein the first reaction chamber orchannel and the first reagent introduction channel are substantiallysealed.
 39. The method of claim 28, wherein the material transportsystem is an electrokinetic control device.
 40. The method of claim 1,the method further comprising selecting a third reagent or mixture ofreagents based upon the results of the analysis of the second reactionproduct, introducing the third reagent or mixture of reagents and athird sample material into the first reaction chamber or channel, or,optionally, into the second reaction chamber or channel, or, optionally,into a third reaction chamber or channel in the microfluidic device,whereupon the third sample material and the third reagent or mixture ofreagents react; and, analyzing a third reaction product.
 41. The methodof claim 28, wherein a plurality of reagents and materials areseparately reacted in the first reaction chamber or channel, the firstreactant and first material being members of the plurality of reagentsand materials, wherein results of a plurality of analyses of thereactants are used to select the second reagent and second material. 42.The method of claim 28, wherein a plurality of reagents and materialsare separately reacted in the first reaction chamber or channel, thefirst reactant and first material being members of the plurality ofreagents and materials, wherein results of a plurality of analyses ofthe reactants are used to select the second reagent and second material,wherein at least 10 different reagents are separately reacted with atleast 10 different materials.
 43. The method of claim 28, comprisingrepeating steps (b)-(g) at least 10 times in the microfluidic deviceprovided in step (a).
 44. The method of claim 28, comprising repeatingsteps (b)-(g) at least 100 times in the microfluidic device provided instep (a).
 45. The method of claim 28, comprising repeating steps (b)-(g)at least 1000 times in the microfluidic device provided in step (a). 46.The method of claim 28, wherein the at least first reactant is a baseand the second reactant is an acid.
 47. The method of claim 28, whereinthe first channel is transverse to a plurality of additional channelsincluding the second channel and in fluid communication with theadditional channels, wherein an aliquot of the first reagent isintroduced into the plurality of additional channels, thereby providinga conversion of serial first reagent flow into parallel first reagentflow.
 48. A method of performing a fluidic operation that requires aplurality of iterative, controlled volume. fluid manipulations, themethod comprising: (a) providing a microfluidic device which includes areaction chamber or channel, a source of a first fluid reactant and asource of a second fluid reactant, a first channel fluidly connectingthe reaction chamber or channel with the source of first fluid reactant,a second channel fluidly connecting the reaction chamber or channel withthe source of second fluid reactant, and a fluid direction system fortransporting preselected volumes of the at least first and second fluidreactants from the source of first fluid reactant or the source ofsecond fluid reactant, respectively, through the first and secondchannels to the reaction chamber or channel; (b) transporting a firstpreselected volume of the at least first fluid reactant to the reactionchamber or channel which first preselected volume is within about 10% ofa first desired volume; (c) transporting a second preselected volume ofthe at least second fluid reactant to the reaction chamber or channel,which second preselected volume is within about 10% of a second desiredvolume; (d) repeating at least one of the steps of transporting thefirst preselected volume of the first fluid reactant or the secondpreselected volume of the second fluid reactant to the reaction chamberor channel.
 49. The method of claim 48, comprising repeating at leastone of the steps of transporting the first preselected volume of thefirst fluid reactant or the second preselected volume of the secondfluid reactant to the reaction chamber or channel at least 10 times. 50.The method of claim 48, wherein the first and second preselected volumesare within about 5% of a first and second desired volume, respectively.51. The method of claim 48, wherein the first and second preselectedvolumes are within about 1% of a first and second desired volume,respectively.
 52. The method of claim 48, wherein at least one of thefirst and second preselected volumes is less than about 1 μl.
 53. Themethod of claim 48, wherein at least one of the first and secondpreselected volumes is less than about 0.1 μl.
 54. The method of claim48, wherein at least one of the first and second preselected volumes isless than about 10 nl.
 55. The method of claim 48, wherein the fluiddirection system is an electroosmotic fluid direction system.
 56. Amethod of optimizing a chemical reaction, comprising: providing amicrofluidic device which includes a reaction chamber or channel, asource of at least a first fluid reactant, a source of at least a secondfluid reactant, and a fluid direction system for delivering a selectedvolume of the first and second reactants to the reaction chamber orchannel; delivering a selected volume of the first reactant to thereaction chamber or channel; delivering a selected volume of the secondreactant to the reaction chamber or channel; mixing the first reactantand the second reactant and incubating the mixed reactants for aselected time; detecting a product of a reaction between the first andsecond reactant; repeating the steps of delivering the first and secondreactants to the reaction chamber or channel, mixing the first andsecond reactants, incubating the mixed reactants and detecting theproduct, wherein at least one selected parameter selected from the groupconsisting of the selected volume of the first reactant, the selectedvolume of the second reactant and the selected incubation time is variedas the steps are repeated; and, determining an optimal level for theselected volume of the first reactant, the selected volume of the secondreactant, or the selected incubation time for producing the product. 57.The method of claim 56, wherein the selected parameter is variedsystematically.
 58. The method of claim 56, wherein the fluid directionsystem is an electrokinetic fluid direction system.
 59. The method ofclaim 56, wherein the optimal level is determined by serially testingthe effect of systematically varying the at least one selected parameterin successive mixing experiments.
 60. The method of claim 56, whereinthe optimal level is determined by testing the effect of changing the atleast one selected parameter in parallel mixing experiments.
 61. Themethod of claim 56, wherein the results of a first mixing experiment areused to select the at least one selected parameter.
 62. The method ofclaim 56, wherein the first and second reactant are mixed at a selectedtemperature.
 63. The method of claim 56, wherein the first and secondreactant are separately mixed at multiple selected temperatures and theoptimal temperature for reaction is determined.
 64. The method of claim63, wherein the temperature is varied systematically.
 65. The method ofclaim 63, wherein the temperature is serially varied in successivemixing experiments.
 66. The method of claim 63, wherein the temperatureis varied in parallel mixing experiments.
 67. The method of claim 56,wherein the reaction chamber or channel is maintained at a selectedtemperature.
 68. The method of claim 56, wherein the first and secondreactant are mixed at a selected pH.
 69. The method of claim 56, whereinthe first and second reactant are separately mixed at multiple selectedpH and the optimal pH for reaction is determined.
 70. The method ofclaim 69, wherein the pH is varied systematically.
 71. The method ofclaim 69, wherein the pH is serially varied in successive mixingexperiments.
 72. The method of claim 69, wherein the pH is varied inparallel mixing experiments.
 73. A system for optimizing and performinga desired chemical reaction, comprising: a microfluidic device whichincludes a reaction chamber or channel, a source of a first reactant anda source of at least a second reactant, fluidly connected to thereaction chamber or channel; an electrokinetic fluid direction systemfor transporting a selected volume of the first reactant to the reactionchamber or channel; a detection system for detecting a result of thechemical reaction; a control system for instructing the fluid directionsystem to deliver a first selected volume of first reactant and a firstselected volume of second reactant to the reaction chamber or channelfor mixing, which mixing produces a first chemical reaction, instructingthe fluid direction system to deliver a second selected volume of firstreactant and a second selected volume of the second reactant to thereaction chamber or channel for mixing, which mixing produces a secondchemical reaction to produce a second chemical reaction, wherein thesecond selected volume of first reactant is optionally varied from thefirst selected volume of first reactant.
 74. The system of claim 73,wherein the control system comprises a computer.
 75. The system of claim73, wherein the microfluidic device includes an element selected fromthe group consisting of a temperature control element for controllingtemperature of reaction of the first and second element, a source ofacid, and a source of base.
 76. The system of claim 73, wherein thecontrol system controls an element of reaction of the first and secondreactant selected from the group consisting of temperature, pH, andtime.
 77. The system of claim 76, wherein the control system directs aplurality of mixings of the first and second reactant, wherein areaction condition selected from the group consisting of temperature,pH, and time is systematically varied in separate mixings.
 78. A methodof performing a fluidic operation that comprises a plurality of parallelfluid manipulations to provide parallel fluidic analysis of samplematerials, the method comprising: providing a microfluidic devicecomprising at least a first transverse reagent introduction channelfluidly connected to a source of at least one reagent and a source of atleast one sample material, the transverse channel fluidly connected to aplurality of parallel reagent reaction channels; selecting a firstreagents from the source of at least one reagent, transporting the firstreagent through the reagent introduction channel and aliquoting aportion of the reagent into at least one parallel reagent reactionchannel; selecting a first sample materials from the source of at leastone sample material and aliquoting the first sample material into atleast a first of the plurality of parallel reagent reaction channels;selecting at least one additional sample material, or at least oneadditional reagent, and aliquoting the additional sample material oradditional reagent into at least a second of the plurality of parallelreagent reaction channels; contacting the first sample material and thefirst reagent in the first reagent reaction channels, whereupon thefirst sample material and the first reagent reacts; contacting the atleast one additional sample material or at least one additional reagentwith one or more fluid component selected from the group consisting ofthe first sample material, the first reagent, the at least oneadditional reagent, the at least one additional sample material, asecond additional reagent, and a second additional sample material;detecting a first reaction product of the first sample material and thefirst reagent; detecting a second reaction product of the at least oneadditional sample material or at least one additional reagent and one ormore fluid component; based upon the first or second reaction product,selecting a secondary reagent and a secondary sample material;introducing the secondary reagent into one of the parallel reactionchannels, whereupon the secondary sample material and the secondaryreagent reacts; and, detecting a secondary reaction product of thesecondary sample material and the secondary reagent, thereby providing afluidic analysis of the first sample material and the secondary samplematerial.
 79. The method of claim 78, wherein the microfluidic devicecomprises an electrokinetic fluid direction system for moving fluidiccomponents in the device.
 80. The method of claim 78, wherein the methodcomprises parallel analysis of a plurality of sample materials in theparallel channels, in which multiple reagents are mixed in a pluralityof the parallel channels with multiple sample materials to form amultiple of products, and, based upon detection of the multipleproducts, selecting the secondary sample material and secondary reagent.81. The method of claim 78, wherein the method comprises parallelanalysis of a plurality of sample materials in the parallel channels, inwhich multiple reagents are mixed in a plurality of the parallelchannels with multiple sample materials to form a multiple of products,and, based upon detection of the multiple products, selecting multiplesecondary sample materials and multiple secondary reagents which aremixed to form multiple reaction products.
 82. The method of claim 80,the method comprising parallel analysis of a plurality of samplematerials and a plurality of reagents in the parallel channels, in whichthe plurality of reagents is mixed in a plurality of the parallelchannels with a plurality of sample materials to form a plurality ofproducts, and, based upon the plurality of products, selecting aplurality of additional sample materials and additional reagents forsubsequent parallel mixing in the parallel channels.
 83. The method ofclaim 78, wherein the microfluidic device includes the first transversereagent introduction channel and at least a second transverse channel,and a plurality of parallel channels intersecting both of the first andsecond transverse channels; and the step of aliquoting the portion ofthe reagent into at least one parallel reagent reaction channel isperformed by: applying a first voltage across the first transversereagent introduction channel and the second transverse channel to drawthe portion of the reagent into the first transverse reagentintroduction channel, whereby the portion of the reagent is present atintersections of the first channel and each of the plurality of parallelchannels; and, applying a second voltage from the first transversechannel to the second transverse channel, whereby a current in each ofthe parallel channels is equivalent, and whereby the portion of thereagent at the intersections of the first transverse channel and each ofthe plurality of parallel channels is moved in to each of the pluralityof parallel channels.
 84. A method of performing a plurality of separateassays on a single sample, comprising: providing a microfluidic devicehaving at least a first transverse channel fluidly connected to at leasta source of the sample, a plurality of separate parallel channelsfluidly connected to the first transverse channel, each of the separatechannels having disposed therein reagents for performing a differentdiagnostic assay, and a fluid direction system for concurrentlydirecting a portion of the sample into each of the plurality of parallelchannels; transporting a portion of the sample into each of the parallelchannels, whereby the sample and the reagents disposed in the channelundergo a reaction; detecting a result of the reaction of the sample andthe reagents disposed in the channel, for each of the parallel channels.85. A method of identifying the presence or absence of a plurality ofdifferent predetermined sequence variations at different loci on atarget nucleic acid sequence, comprising: delivering the target nucleicacid sequence to each of a plurality of separate reaction chamber orchannels; in each of the reaction chamber or channels, amplifyingseparate regions of the target nucleic acid sequence, each of theseparate regions encompassing at least one of the different loci;determining whether each of the separate regions contains the sequencevariation.
 86. The method of claim 85, wherein the step of determiningcomprises determining a size of the separate region and comparing it toa size of the separate region in the absence of the sequence variation,a difference being indicative of a sequence variation in the region. 87.A method of performing a fluidic operation that requires a plurality ofsuccessive fluid manipulations on a sample, comprising: providing amicrofluidic device that comprises at least a first channel fluidlyconnected to a source of the sample, the first channel being intersectedby at least second and third channels, the second and third channelsbeing fluidly connected to a source of first fluid reactant and a sourceof second fluid reactant, respectively; transporting a volume of thesample from the source of sample into the first channel; transporting avolume of the first fluid reactant from the source of first fluidreactant to the first channel to combine with the sample; andtransporting a volume of the second fluid reactant from the source ofsecond fluid reactant to the first channel to combine with the sample.88. The method of claim 87, wherein the device comprises anelectrokinetic fluid direction system and the transporting steps areperformed by electrokinesis.
 89. The method of claim 87, wherein thesample is a nucleic acid, the first fluid reactant comprises asequencing mixture and the second fluid reactant comprises a sequencingstop buffer.
 90. The method of claim 87, wherein the first fluidreactant and second fluid reactant are sequencing reagents.
 91. Themethod of claim 87, wherein a product of the combination of the firstand second reactants and the sample is detected.
 92. The method of claim87, wherein a product of the combination of the first and secondreactants and the sample is detected, wherein results of the detectionare used to select a second sample, or to select an additional reactantfor subsequent mixing with a component selected from the groupconsisting of the first reactant, the second reactant, the first sample,the second sample and the additional reactant.
 93. A microfluidic devicefor concurrently performing a plurality of diagnostic assays on asample, comprising: a plurality of parallel reaction channels, each ofthe reaction channels having disposed therein reagents for performing adifferent diagnostic assay; a source of the sample; a sampleintroduction channel which is fluidly connected to the source of sampleand which intersects each of the parallel channels; a fluid directionsystem for directing a portion of the sample into each of the pluralityof different parallel channels.
 94. The microfluidic device of claim 93,wherein the device further comprises a control element for selectingwhich diagnostic assays are run in the device.
 95. The microfluidicdevice of claim 93, wherein the device further comprises a controlelement for selecting which diagnostic assays are run in the device,wherein the control element selects a first diagnostic assay, detectsthe results of the first assay and selects a second diagnostic assaybased upon the results of the first assay.
 96. The microfluidic deviceof claim 93, wherein the device further comprises a control element forselecting which diagnostic assays are run in the device, wherein thecontrol element selects a first parallel series of diagnostic assays,detects the results of the first parallel series of assays and selects asecond series of diagnostic assays based upon the results of the firstseries of assays.
 97. A microfluidic device for concurrently identifyinggenetic markers at a plurality of different loci on a target nucleicacid sequence, comprising: a plurality of reaction chamber or channels,each of the reaction chamber or channels containing reagents foramplification of a different locus on the target nucleic acid sequence;a source of the target nucleic acid; a sample introduction channelfluidly connecting the source of target nucleic acid and each of thereaction chamber or channels; a plurality of separation channels, eachfluidly connected to a different one of the reaction chamber orchannels; a fluid direction system for transporting a volume of thetarget nucleic acid sequence to each of the reaction chamber or channelsvia the sample channel, and for transporting an amplified product fromthe reaction chamber or channels through each of the separationchannels.
 98. The microfluidic device of claim 97, wherein the devicefurther comprises a joule heating element for PCR amplification of thetarget nucleic acid.
 99. The microfluidic device of claim 97, whereinthe device further comprises at least one source of a plurality ofoligonucleotides, at least one of which is complementary to the targetnucleic acid.
 100. A method of amplifying a target nucleic acid by anon-thermal polymerase chain reaction, the method comprising: providinga microfluidic device which includes a reaction chamber or channelcontaining a target nucleic acid sequence and primer sequences, a sourceof a chemical denaturant and a source of polymerase enzyme fluidlyconnected to the reaction chamber or channel, and a fluid directionsystem for delivering the chemical denaturant or the polymerase enzymeto the reaction chamber or channel; melting complementary strands of thetarget nucleic acid sequence by delivering a volume of the chemicaldenaturant to the reaction chamber or channel; annealing the primersequences to the target nucleic acid by eliminating a denaturing effectof the chemical denaturant; extending the primer sequences along thetarget nucleic acid sequence by delivering a volume of the polymeraseenzyme to the reaction chamber or channel; and repeating the steps ofmelting, annealing and extending to amplify the target nucleic acidsequence.
 101. The method of claim 100, wherein the chemical denaturantis a base, and the annealing step comprises neutralizing the base bydelivering an effective amount of an acid to the reaction chamber orchannel.
 102. The method of claim 100, wherein the base is NaOH and theacid is HCl.
 103. The method of claim 100, wherein the polymerase enzymeis selected from Taq polymerase, Pfu DNAse, Bst and Vent polymerase.104. The method of claim 100, wherein the steps of melting, annealingand extending are repeated from about 10 to about 50 times.
 105. Amicrofluidic device for identifying the presence of a target nucleicacid in a sample, comprising: at least a first reaction channel havingfirst and second termini, the reaction channel having at least first,second and third groups of oligonucleotide probes immobilized in first,second and third regions of the reaction channel, respectively, each ofthe first, second and third probes being complementary to the targetsequence, whereby each of the first second and third probes hybridizesto the target sequence with a different hybridization strength; a sourceof a sample fluidly connected to one of the first and second termini; asource of a denaturant gradient fluidly connected to the first terminus;a fluid direction system for selectively transporting a sample from thesource of sample and a denaturant gradient from the source of denaturantgradient, to the reaction channel.
 106. The microfluidic device of claim105, wherein the first second and third probes bind to the targetsequence with the same avidity.
 107. The microfluidic device of claim105, wherein the first second and third probes bind to the targetsequence with a different avidity.
 108. The microfluidic device of claim105, wherein the fluid direction system is an electrokinetic fluiddirection system.
 109. The microfluidic device of claim 105, wherein thefirst probe binds under stringent conditions to a first allele of atarget nucleic acid, and the second or third probe binds to a second orthird allele of the target nucleic acid.
 110. The microfluidic device ofclaim 105, wherein the first second and third probes bind to differentregions of the target nucleic acid.
 111. The microfluidic device ofclaim 105, wherein the first second and third probes bind to the sameregion of the target nucleic acid.
 112. A microfluidic device foridentifying the presence or absence of a sequence variation in a targetnucleic acid sequence, comprising: a source of the target nucleic acid;a source of oligonucleotide probes that are complementary to an expectedsequence of the target nucleic acid sequence; a source of a chemicaldenaturant; a reaction channel fluidly connected to each of the sourceof target nucleic acid, oligonucleotide probes and chemical denaturant;a fluid direction system for transporting a volume of the target nucleicacid sequence and oligonucleotide probes to the reaction channel, andfor delivering a concentration gradient of the chemical denaturant tothe reaction channel.